Archives of Ophthalmology

Albert, Daniel M.

1998 Archives of Ophthalmology

doi: 10.1001/archopht.116.1.9

Below are the names of reviewers who have graciously and thoughtfully assisted the Editorial Board, our authors, and our readers during the past year. A T. Aaberg, Atlanta, Ga; M. Abelson, North Andover, mMass; G. Abrams, Detroit, Mich; D. Abramson, New York, NY; A. Adamis, Boston, Mass; L. Aiello, Jr, Boston, Mass; M. Albertini, Madison, Wis; R. Allen, Richmond, Va; R. R. Allingham, Durham, NC; J. Alvarado, San Francisco, Calif; D. Anderson, Miami, Fla; R. Anderson, Salt Lake City, Utah; C. J. Anderson, Madison, Wis; B. Anderson, Jr, Durham, NC; D. Apple, Charleston, SC; A. Arnold, Los Angeles, Calif; S. Atherton, San Antonio, Tex; R. Atkinson, Jr, Madison, Wis; J. Auran, New York, NY; D. Azar, Boston, Mass. B G. Baerveldt, Cleveland, Ohio; K. Bailey, Rochester, Minn; A. Bajart, Boston, Mass; E. Balish, Madison, Wis; K. Baratz, Rochester, Minn; N. Barney, Madison, Wis; G. Bartley, Rochester, Minn; J. B. Bateman, Denver, Colo; J. Baum, Wellesley Hills, Mass; R. Bechtel, Altoona, Pa; A. Beck, Atlanta, Ga; G. Beck, Cleveland, Ohio; R. Beck, Tampa, Fla; D. Beecher, Madison, Wis; M. Belin, Albany, NY; M. Belkin, Tel Hashomer, Israel; W. Benson, Wyndmoor, Pa; J. Berestka, Plainfield, Ill; P. Bernstein, Salt Lake City, Utah; A. Biglan, Pittsburgh, Pa; J. Bilyk, Huntingdon Valley, Pa; P. Binder, San Diego, Calif; D. Birch, Dallas, Tex; E. Birch, Dallas, Tex; G. Blankenship, Hershey, Pa; B. Blodi, Madison, Wis; M. Blumenkranz, Menlo Park, Calif; S. Bogan, Shelby, NC; M. Borchert, Los Angeles, Calif; W. Bourne, Rochester, Minn; C. Brandt, Madison, Wis; J. Brandt, Sacramento, Calif; S. Bressler, Baltimore, Md; F. Brightbill, Madison, Wis; R. Brockhurst, Lynnfield, Mass; M. Brodsky, Little Rock, Ark; S. E. Brooks, Augusta, Ga; J. Brown, Iowa City, Iowa; D. Brown, Los Angeles, Calif; G. Brown, Wyndmoor, Pa; S. Brownstein, Ottawa, Ontario; R. Brubaker, Rochester, Minn; H. Buettner, Rochester, Minn; D. Bok, Los Angeles, Calif; D. Budenz, Miami, Fla; C. Burgoyne, New Orleans, La; L. Burk, Dallas, Tex; M. Burnier, Montreal, Quebec; M. Burnstine, Los Angeles, Calif; W. Busse, Madison, Wis; J. Butler, Atlanta, Ga. C D. Callanan, Arlington, Tex; D. Cameron, Minneapolis, Minn; D. Campbell, Lebanon, NH; J. Campbell, Rochester, Minn; P. Campochiaro, Baltimore, Md; C. Camras, Omaha, Neb; J. Caprioli, Los Angeles, Calif; R. Carassa, Milan, Italy; R. Carr, New York, NY; C. Chan, Bethesda, Md; S. Chang, New York, NY; S. Charles, Memphis, Tenn; B. Chauhan, Halifax, Nova Scotia; E. Chew, Bethesda, Md; S. Christiansen, Little Rock, Ark; E. Chuang, Seattle, Wash; L. Chylack, Boston, Mass; G. Cioffi, Portland, Ore; J. Clarkson, Miami, Fla; A. Coleman, Los Angeles, Calif; D. J. Coleman, New York, NY; A. Colenbrander, San Francisco, Calif; M. Conway, New Orleans, La; S. Cousins, Miami, Fla; T. Cox, Chapel Hill, NC; J. Crawford, Toronto, Ontario; A. Crichton, Calgary, Alberta; S. Cross, Rochester, Minn; K. Cruickshanks, Madison, Wis; W. Culbertson, Miami, Fla. D D. D'Alessio, Madison, Wis; R. D'Amico, New York, NY; D. D'Amico, Boston, Mass; C. Dabbs, Toledo, Ohio; S. Daiger, Houston, Tex; R. Dallow, Boston, Mass; R. Danis, Indianapolis, Ind; R. David, Irvine, Calif; J. Davis, Miami, Fla; J. Davis, Madison, Wis; C. Dawson, San Francisco, Calif; E. De Juan, Baltimore, Md; M. del Cerro, Rochester, NY; J. Demer, Los Angeles, Calif; D. Demets, Madison, Wis; L. DeSantis, Fort Worth, Tex; M. Destro, Brookline, Mass; T. Deutsch, Chicago, Ill; J. Diamond, New Orleans, La; K. Dickersin, Baltimore, Md; J. P. Dieckert, Belton, Tex; M. Diener-West, Baltimore, Md; K. Digre, Salt Lake City, Utah; J. Dion, Charlottesville, Va; B. Doft, Pittsburgh, Pa; C. Dohlman, Boston, Mass; S. Donahue, Nashville, Tenn; E. Donnenfeld, Rockville Center, NY; P. Donzis, Marina del Rey, Calif; R. Dortzbach, Madison, Wis; A. Drack, Atlanta, Ga; S. Drance, Vancouver, British Columbia; S. Dresner, Santa Monica, Calif; W. Driebe, Gainesville, Fla; E. Dreyer, Philadelphia, Pa; J. Duker, Boston, Mass; J. Dunn, Baltimore, Md; J. Dutton, Durham, NC. E-F R. Eagle, Philadelphia, Pa; M. Easterbrook, Toronto, Ontario; E. Ebert, Richmond, Va; H. Edelhauser, Atlanta, Ga; J. Edelman, Irvine, Calif; J. Egbert, Minneapolis, Minn; D. Elder, Philadelphia, Pa; S. Elner, Ann Arbor, Mich; J. Emery, Houston, Tex; R. Engerman, Madison, Wis; D. Epstein, Durham, NC; J. T. Ernest, Chicago, Ill; M. Farber, Delmar, NY; R. Fechtner, Louiville, Ky; J. Federman, Winnewood, Pa; S. Fekrat, Baltimore, Md; S. Feldon, Los Angeles, Calif; T. Ferguson, St Louis, Mo; F. Ferris, Bethesda, Md; R. Fine, Boston, Mass; S. Fine, Philadelphia, Pa; D. Finkelstein, Baltimore, Md; M. Fisher, Madison, Wis; G. Fishman, Chicago, Ill; A. Flach, Corte Madera, Calif; R. Flower, Towson, Md; H. Flynn, Miami, Fla; R. Folberg, Iowa City, Iowa; J. Folk, Iowa City, Iowa; D. Fong, Los Angeles, Calif; R. Font, Houston, Tex; R. Forster, Miami, Fla; C. S. Foster, Boston, Mass; T. France, Madison, Wis; R. Frank, Bloomfield Hill, Mich; F. Fraunfelder, Portland, Ore; T. Freddo, Boston, Mass; W. Freeman, La Jolla, Calif; T. Friberg, Pittsburgh, Pa; E. Friedman, Boston, Mass; L. Frohman, Newark, NJ; D. Fryback, Madison, Wis; A. Fulton, Boston, Mass; J. Funderburgh, Manhattan, Kan. G B. Gallie, Toronto, Ontario; J. Gamel, Louisville, Md; G. Garcia, Brookline, Mass; T. Gardner, Hershey, Pa; J. Garrity, Rochester, Minn; J. D. Gass, Nashville, Tenn; R. Gausas, Philadelphia, Pa; B. Geiger, Rehovot, Israel; L. Gentry, Madison, Wis; S. Gieser, Baltimore, Md; J. Gilbard, Boston, Mass; W. Gilbert, Chevy Chase, Md; H. Gimbel, Calgary, Alberta; I. Gipson, Boston, Mass; B. Glaser, Baltimore, Md; J. Glaser, Miami, Fla; B. Glasgow, Los Angeles, Calif; R. Glynn, Boston, Mass; M. Goldberg, Baltimore, Md; R. Gonnering, Milwaukee, Wis; L. Gordon, Los Angeles, Calif; M. Gordon, St. Louis, Mo; M. Goren, Chicago, Ill; J. Gottlieb, Madison, Wis; J. Gottsch, Baltimore, Md; P. Gouras, New York, NY; E. Gragoudas, Boston, Mass; K. Green, Augusta, Ga; D. Greenfield, New York, NY; F. Greer, Madison, Wis; C. Grosskreutz, Boston, Mass; H. Grossnicklaus, Atlanta, Ga; A. Grove, Boston, Mass; F. Gutman, Cleveland, Ohio; D. Guyer, New York, NY; D. Guyton, Baltimore, Md. H W. Hagler, Atlanta, Ga; W. Halfter, Pittsburgh, Pa; J. Haller, Baltimore, Md; L. Hamed, Gainesville, Fla; D. Han, Milwaukee, Wis; D. Hardten, Minneapolis, Minn; C. Hardwick, Birmingham, Ala; P. Hargrave, Gainesville, Fla; A. Harris, Indianapolis, Ind; D. Harris, Knoxville, Tenn; G. Harris, Milwaukee, Wis; T. Harrison, Anchorage, Alaska; S. Hayreh, Iowa City, Iowa; L. Hazlett, Detroit, Mich; J. Heckenlively, Los Angeles, Calif; T. Hedges, Boston, Mass; R. Hemady, Baltimore, Md; E. Heon, Toronto, Ontario; D. Herman, Rochester, Minn; M. Hettinger, Overland Park, Kan; D. Heuer, Los Angeles, Calif; A. Hidayat, Rockville, Md; R. Hill, Irvine, Calif; A. Hillis, Temple, Tex; D. Hinton, Los Angeles, Calif; P. Hersh, New York, NY; R. Hitchings, London, EnglandK. Hoffer, Santa Monica, Calif; N. Holekamp, St. Louis, Mo; J. Holladay, Houston, Tex; E. Holland, Minneapolis, Minn; G. Holland, Los Angeles, Calif; P. Holm, Chippewa Falls, Wis; J. Holmes, Rochester, Minn; A. Hornblass, New York, NY; H. D. Hoskins, San Francisco, Calif; M. Lynch, Atlanta, Ga; E. Howes, San Francisco, Calif; C. Hoyt, San Francisco, Calif; W. Hoyt, San Francisco, Calif; S. Huang, Cleveland, Ohio; A. Huang, Miami, Fla; A. Huang, Minneapolis, Minn; J. Hunkeler, Kansas City, Mo; D. Hunter, Baltimore, Md; A. Huntley, Sacramento, Calif; J. Hurwitz, Toronto, Ontario; B. T. Hutchinson, Boston, Mass. I-J G. Inana, Miami, Fla; S. Isenberg, Los Angeles, Calif; D. Jabs, Baltimore, Md; S. Jacobson, Philadelphia, Pa; D. Jacobson, Marshfield, Wis; G. Jaffe, Durham, NC; L. Jameson, Madison, Wis; H. Jampel, Baltimore, Md; N. Janz, Ann Arbor, Mich; W. Jarrett, Atlanta, Ga; K. Johns, Nashville, Tenn; M. Johnson, Ann Arbor, Mich; C. Johnson, Sacramento, Calif; J. Jonas, Erlangen, Germany; K. Joos, Nashville, Tenn; N. Joyce, Boston, Mass. K R. Kalina, Seattle, Wash; H. Kaplan, St. Louis, Mo; R. Kardon, Iowa City, Iowa; M. Kass, St Louis, Mo; J. Katz, Baltimore, Md; B. Katz, Rochester, NY; H. Kaufman, New Orleans, La; P. Kaufman, Madison, Wis; A. Kaufman, Cincinnati, Ohio; J. Keane, Los Angeles, Calif; R. Keech, Iowa City, Iowa; U. Keesey, Madison, Wis; S. Kelnan, Baltimore, Md; S. Kelsey, Pittsburgh, Pa; J. Keltner, Davis, Calif; J. Kennerdell, Pittsburgh, Pa; K. Kenyon, Boston, Mass; T. Kern, Cleveland, Ohio; B. Keyser, Cranbury, NJ; S. Khwarg, Torrance, Calif; D. Kikkawa, La Jolla, Calif; A. Kimura, Iowa City, Iowa; B. Klein, Madison, Wis; R. Klein, Madison, Wis; L. Kline, Birmingham, Ala; G. Klintworth, Durham, NC; S. Klyce, New Orleans, La; D. Knox, Baltimore, Md; D. Koch, Houston, Tex; A. Kolker, St Louis, Mo; V. Kowal, Rapid City, SD; J. Krachmer, Minneapolis, Minn; T. Kramer, Tucson, Ariz; H. Krauss, Santa Monica, Calif; J. Kronish, Delray Beach, Fla; R. Krueger, St Louis, Mo; T. Krupin, Chicago, Ill; M. Kupersmith, New York, NY; B. Kupperman, Irvine, Calif; Y. Kwon, Iowa City, Iowa. L P. Laibson, Philadelphia, Pa; B. Lam, Miami, Fla; H. M. Lambert, Houston, Tex; S. Lambert, Atlanta, Ga; M. Landers, Virginia Beach, Va; D. Pavan Langston, Boston, Mass; A. Laties, Philadelphia, Pa; N. Laughlin, Madison, Wis; M. LaVail, San Francisco, Calif; J. Leavitt, Rochester, Minn; C. Lederer, Kanasas City, Mo; A. Lee, Houston, Tex; D. Lee, Los Angeles, Calif; H. Leibowitz, Boston, Mass; B. Lemke, Madison, Wis; M. Lemp, Washington, DC; C. Leone, Jr, San Antonio, Tex; M. Lesk, Montreal, Quebec; M. C. Leske, Stonybrook, NY; R. Lesser, Waterbury, Conn; L. Levi, La Jolla, Calif; M. Levin, Highland Park, Ill; M. Lewis, Miami, Fla; R. Lewis, Sacramento, Calif; R. A. Lewis, Houson, Tex; M. Lieberman, San Francisco, Calif; J. Liebmann, New York, NY; T. Liesegang, Jacksonville, Fla; J. Lilien, Detroit, Mich; J. Lim, San Francisco, Calif; T. Lindquist, Seattle, Wash; D. Litoff, Greenwich, Conn; C. Lobeck, Middleton, Wis; P. Lopez, Miami, Fla; B. Lotz, Madison, Wis; M. Lucarelli, Madison, Wis; G. Lutty, Baltimore, Md; M. Luxenberg, Augusta, Ga; D. Lyon, Kansas City, Mo. M R. Machemer, Durham, NC; R. MacKool, Astoria, NY; S. Macrae, Portland, Ore; M. Macsai, Morgantown, WVa; S. Madreperla, Cleveland, Ohio; L. Magargal, Philadelphia, Pa; L. Maguire, Rochester, Minn; A. Maguire, Philadelphia, Pa; M. Maguire, Philadelphia, Pa; M. Mainster, Kansas City, Kan; N. Mamalis, Salt Lake City, Utah; M. Mandel, Hayward, Calif; C. Mangione, Los Angeles, Calif; M. Mannis, Sacramento, Calif; D. Marcus, Augusta, Ga; J. Mares-Perlman, Madison, Wis; C. Margo, Tampa, Fla; T. Margolis, San Francisco, Calif; M. Marmor, Stanford, Calif; D. Martin, Atlanta, Ga; W. Mathers, Iowa City, Iowa; C. Mattox, Boston, Mass; D. Maurice, New York, NY; L. Mayer, Boston, Mass; I. McAllister, Nedlands, Australia; C. McCord, Jr, Atlanta, Ga; C. McKeown, Boston, Mass; S. McKinnon, Baltimore, Md; I. McLean, Washington, DC; M. Mead, Boston, Mass; R. Meckler, Louisville, Ky; C. Mehta, Madison, Wis; D. Meisler, Cleveland, Ohio; G. Mejicano, Madison, Wis; S. Melamed, Tel Hashomer, Israel; N. Melberg, St Louis, Mo; B. M. Melia, Baltimore, Md; T. Meredith, St Louis, Mo; M. Mets, Chicago, Ill; H. Metz, Rochester, NY; R. Meyer, Ann Arbor, Mich; F. Mikelberg, Vancouver, British Columbia; J. Miller, Boston, Mass; M. Miller, Chicago, Ill; N. Miller, Baltimore, Md; M. Mills, Madison, Wis; R. Mills, Seattle, Wash; J. Mims III, San Antonio, Tex; D. Minckler, Los Angeles, Calif; T. Mittag, New York, NY; H. Moffet, Madison, Wis; B. Mondino, Los Angeles, Calif; L. Morse, Sacramento, Calif; S. Moscella, Burlington, Mass; A. Moser, Madison, Wis; M. Moster, Philadelphia, Pa; C. Moy, Baltimore, Md; S. Mukai, Boston, Mass; F. Munier, Lusanne, Switzerland; R. Murphy, Baltimore, Md; T. Murray, Miami, Fla. N-O J. D. Nelson, St Paul, Minn; J. Nerad, Iowa City, Iowa; P. Netland, Riyadh, Saudi Arabia; N. Newman, Atlanta, Ga; S. Newman, Charlottesville, Va; C. Newton, Louisville, Ky; D. Nichols, St Paul, Minn; R. Nickelis, Madison, Wis; M. Nicolela, Vancouver, British Columbia; J. Niederkorn, Dallas, Tex; T. M. Nork, Madison, Wis; R. Nozik, Lafayette, Calif; W. Nunery, Indianapolis, Ind; R. Nussenblatt, Bethesda, Md; P. O'Brien, Rochester, Minn; T. O'Brien, Baltimore, Md; J. O'Brien, San Francisco, Calif; D. O'Day, Nashville, Tenn; R. J. Olk, St Louis, Mo; T. Olsen, Madison, Wis; N. Osborne, Oxford, England; A. Ottlecz, Houston, Tex; C. Owsley, Birmingham, Ala. P-Q A. Palestine, Washington, DC; P. Palmberg, Miami, Fla; J. Parel, Miami, Fla; S. Park, Dallas, Tex; R. Parrish, Miami, Fla; J. Patrinely, Houston, Tex; M. Pavilack, Lancaster, Pa; C. Pavlin, Toronto, Ontario; D. Pavan Langston, Boston, Mass; W. Pearce, Edmonton, Alberta; K. Peele, Bethesda, Md; R. Peiffer, Jr, Chapel Hill, NC; J. Pepose, St Louis, Mo; D. Peters, Madison, Wis; G. Peyman, New Orleans, La; R. Pfister, Birmingham, Ala; S. Pflugfelder, Miami, Fla; G. Pier, Boston, Mass; E. Pierce, Boston, Mass; A. R. Pilkerton, Jr, Chevy Chase, Md; S. Podos, New York, NY; J. Pokorny, Chicago, Ill; A. Polans, Madison, Wis; M. Powers, Portland, Ore; M. Preslan, Baltimore, Md; F. Price, Indianapolis, Ind; R. Proctor, Madison, Wis; R. Pruett, Boston, Mass; C. Puliafito, Boston, Mass; J. Pulido, Milwaukee, Wis; J. Purcell, St Louis, Mo; V. Purvin, Indianapolis, Ind; H. Quigley, Baltimore, Md. R Y. Rabinowitz, Los Angeles, Calif; N. Rao, Los Angeles, Calif; P. Rapoza, Boston, Mass; C. Rapuano, Philadelphia, Pa; B. Reese, Santa Barbara, Calif; M. Refojo, Boston, Mass; T. Reid, Davis, Calif; R. Reinecke, Philadelphia, Pa; G. Reizner, Madison, Wis; M. Repka, Baltimore, Md; D. Rickman, Coralville, Iowa; R. Rieselbach, Madison, Wis; M. Riley, Rochester, Mich; R. Ritch, New York, NY; M. Rizzo, Iowa City, Iowa; R. Robb, Boston, Mass; D. Robertson, Rochester, Minn; A. Robin, Baltimore, Md; J. Robin, Cleveland, Ohio; W. Robison, Bethesda, Md; J. Rootman, Vancouver, British Columbia; J. Rosenbaum, Portland, Ore; L. Rosenberg, Chicago, Ill; G. O. Rosenwasser, Hershey, Pa; W. Rowley, Ames, Iowa; S. Roy, Boston, Mass; G. Rubin, Baltimore, Md; M. Rubin, Gainesville, Fla; P. Rubin, Boston, Mass; P. Rutecki, Madison, Wis; M. Ruttum, Milwaukee, Wis; S. Ryan, Los Angeles, Calif. S A. Sadun, Los Angeles, Calif; D. Schanzlin, St Louis, Mo; H. Schatz, San Francisco, Calif; N. Schatz, Miami, Fla; O. Schein, Baltimore, Md; M. Schluchter, Cleveland, Ohio; E. Schmidt, Bethesda, Md; S. Schocket, Owings Mills, Md; J. Schuman, Boston, Mass; R. Schumer, New York, NY; B. Schwartz, Boston, Mass; W. Scott, Iowa City, Iowa; J. Seddon, Boston, Mass; D. Seigel, Cushing, Me; R. Sergott, Philadelphia, Pa; S. Shapiro, Madison, Wis; S. Sharma, Valhalla, NY; V. Sheffield, Iowa City, Iowa; N. Sher, Minneapolis, Minn; D. Sherman, Nashville, Tenn; M. Sherwood, Gainesville, Fla; D. Shetlar, Nashville, Tenn; C. Shields, Philadelphia, Pa; J. Shields, Philadelphia, Pa; D. Shin, Detroit, Mich; B. Shingleton, Boston, Mass; J. Shore, Austin, Tex; W. T. Shults, Portland, Ore; P. Sibony, Stony Brook, NY; P. Sieving, Ann Arbor, Mich; D. Silver, Laurel, Md; D. Silverstone, New Haven, Conn; R. Simmons, Boston, Mass; L. Singerman, Beachwood, Ohio; B. Sires, Seattle, Wash; G. Skuta, Oklahoma City, Okla; M. Slavin, New Hyde Park, NY; K. Small, Los Angeles, Calif; W. Smiddy, Miami, Fla; L. Smith, Boston, Mass; M. Smith, Madison, Wis; R. Smith, Los Angeles, Calif; S. Sneed, Mesa, Ariz; R. Snyder, Tucson, Ariz; A. Sober, Boston, Mass; K. Solomon, Charleston, SC; A. Sommer, Baltimore, Md; K. Soong, Ann Arbor, Mich; S. Soukiasian, Watertown, Mass; G. Spaeth, Philadelphia, Pa; M. Speaker, New York, NY; W. Spencer, San Francisco, Calif; R. Sperduto, Bethesda, Md; B. Spivey, Chicago, Ill; D. Stager, Dallas, Tex; R. Stamper, San Francisco, Calif; W. Stark, Baltimore, Md; R. Steeves, Madison, Wis; R. Steinert, Boston, Mass; S. Stenson, New York, NY; P. Sternberg, Atlanta, Ga; W. Stewart, Charleston, SC; R. Stone, Philadelphia, Pa; B. Streeten, Syracuse, NY; G. Striph, Toledo, Ohio; C. Strother, Madison, Wis; R. Stulting, Atlanta, Ga; A. Sugar, Ann Arbor, Mich; J. Sugar, Chicago, Ill; A. Baker, Boston, Mass; N. Syed, Philadelphia, Pa. T J. Talamo, Boston, Mass; K. Tarbet, Madison, Wis; W. Tasman, Philadelphia, Pa; M. Terry, Portland, Ore; C. Thirkill, Sacramento, Calif; M. Thomas, St Louis, Mo; H. S. Thompson, Iowa City, Iowa; J. Thompson, Towson, Md; K. Thompson, Atlanta, Ga; J. Tiedeman, Charlottesville, Va; J. Tielsch, Baltimore, Md; D. Tingey, London, Ontario; F. Tolentino, Boston, Mass; R. Tomsak, Cleveland, Ohio; T. Topping, Boston, Mass; C. Toris, Omaha, Neb; C. Toth, Durham, NC; D. Townsend, Boston, Mass; E. Traboulsi, Baltimore, Md; B. Tripathi, Columbia, SC; R. Tripathi, Columbia, SC; J. Trobe, Ann Arbor, Mich; S. Trocme, Galveston, Tex; S. Trokel, New York, NY; D. Tse, Miami, Fla; S. Tseng, Miami, Fla; J. Turner, Winston-Salem, NC; P. Turski, Madison, Wis; S. Twining, Milwaukee, Wis. U-W S. Unterman, Kansas City, Mo; E. M. Van Buskirk, Portland, Ore; R. Varma, Los Angeles, Calif; T. Verstraeten, Pittsburgh, Pa; N. Volpe, Philadelphia, Pa; M. Vrabec, Appleton, Wis; M. Wall, Iowa City, Iowa; R. Waller, Rochester, Minn; D. Walton, Boston, Mass; M. Wand, Hartford, Conn; G. Waring, Atlanta, Ga; D. Watt, Annapolis, Md; M. Wax, St Louis, Mo; D. Weinberg, Santa Monica, Calif; T. Weingeist, Iowa City, Iowa; J. Weinstein, Middleton, Wis; H. Weiss, Washington, DC; J. Weiss, Detroit, Mich; J. Weisz, Baltimore, Md; J. Weiter, Boston, Mass; R. Welch, Baltimore, Md; R. Weleber, Portland, Ore; S. West, Baltimore, Md; C. Weyand, Rochester, Minn; S. Whitcup, Bethesda, Md; V. White, Vancouver, British Columbia; P. Wiedemann, Leipzig, Germany; J. Wiggs, Boston, Mass; J. Wilensky, Chicago, Ill; K. Wilhelmus, Houston, Tex; G. Williams, Royal Oak, Mich; J. K. Willson, Cleveland, Ohio; D. Wilson, Portland, Ore; F. Wilson, Indianapolis, Ind; S. Wilson, Cleveland, Ohio; J. Windle, San Antonio, Tex; M. Wolff, Madison, Wis; D. Wolfley, New Haven, Conn; F. Wong, Durham, NC; J. Woog, Boston, Mass; K. Wright, Cleveland, Ohio; G. Wu, Brookline, Mass. Y-Z M. Yablonski, Omaha, Neb; L. Yannuzzi, New York, NY; M. Yanoff, Philadelphia, Pa; R. Yee, Indianapolis, Ind; L. Young, Boston, Mass; D. Zack, Baltimore, Md; L. Zangwill, La Jolla, Calif; D. Zee, Baltimore, Md; L. Zimmerman, Washington, DC; T. Zimmerman, Louisville, Ky; G. Zinser, Heidelberg, Germany; J. Zwaan, San Antonio, Tex.

Ghaith, Alaa A.; Daniel, Jan; Stulting, R. Doyle; Thompson, Keith P.; Lynn, Michael

1998 JAMA Ophthalmology

doi: 10.1001/archopht.116.1.12pmid: 9445203

ObjectivesTo study the effects of radial keratotomy (RK) and photorefractive keratectomy (PRK) on contrast sensitivity and glare disability using 4 different devices, and to correlate subjective complaints with objective scores of visual performance.MethodsPreoperative contrast sensitivity for 30 eyes undergoing RK and 30 eyes undergoing PRK was compared with contrast sensitivity at 1, 3, and 6 months postoperatively using the CSV 1000, MCT (Multivision Contrast Tester) 8000, and Pelli-Robson chart. The BAT (Brightness Acuity Tester) and MCT 8000 were used to test for daytime and nighttime glare disability, respectively. At 3 and 6 months postoperatively, a questionnaire was administered to assess visual performance subjectively.ResultsContrast sensitivity decreased after RK and PRK up to the sixth postoperative month, while glare disability was significantly increased at 1 month after PRK as determined by the MCT 8000 and the BAT, and at the third and sixth months after RK using the MCT 8000. Compared with RK, PRK significantly decreased contrast sensitivity as measured with the MCT 8000 at all spatial frequencies 1 month postoperatively. No significant difference in visual performance between patients undergoing RK and PRK was observed with the CSV 1000, the Pelli-Robson chart, or the BAT up to 6 months postoperatively. No consistent difference was found between glare disability scores of patients undergoing RK and PRK when measured with the MCT 8000. Subjective reports of problems with night driving and blurring correlated only with glare disability scores of the MCT 80003 months after RK.ConclusionsBoth RK and PRK reduce contrast sensitivity and cause glare disability; however, the relative effect is highly dependent on the time postoperative testing is performed and the instrument used for testing. Contrast sensitivity and glare disability, as measured by the instruments used in this study, do not accurately reflect patients' subjective assessment of visual performance in daily life.MOST CLINICAL studies assess the safety of refractive surgical procedures by measuring the change in best spectacle-corrected visual acuity. Visual acuity, however, is a crude measure of visual performance. Visual acuity is usually measured by determining a person's ability to resolve fine spatial detail using high-contrast targets (black figures on a white background), such as Snellen letters, numbers, or Landolt C rings. The contrast level of these targets approaches 100%. In everyday life, however, such high-contrast targets are rarely encountered, and patients who score well on traditional visual acuity tests may complain of poor vision in everyday situations.Such patients are believed to have decreased contrast sensitivity (CS), which impairs their ability to identify objects with low contrast under low lighting conditions.Contrast sensitivity is defined as the ability to detect differences in luminance between adjacent areas. Contrast sensitivity tests determine the threshold of contrast required to identify a target.The target may be letters or sine wave gratings. The latter, which are most widely used, consist of alternating dark and light bars. These bars are specified according to their size (spatial frequency), contrast, and orientation. The CS function is a curve produced by plotting the minimum or threshold contrast (on the y-axis) required to identify a target against spatial frequency of the target (on the x-axis).Standard visual acuity measurement does not adequately test visual performance under bright indirect lighting conditions such as that of a bright sunny day or headlights from an oncoming car. A patient with partial opacification of the ocular media (eg, corneal scars, corneal haze, or lenticular opacities)may suffer from glare disability (GD), which is the reduction of visual acuity caused by light elsewhere in the field of vision.Opacities of the ocular media cause intraocular light scattering, which interferes with perception of the object of regard.Glare disability is generally tested by measuring vision with and without the addition of a light source located away from the object of regard.The terms glareand glare disability(GD) will be used interchangeably throughout this report.The effects of radial keratotomy (RK) and photorefractive keratectomy (PRK) on CS and GD have been previously investigated.However, most of these studies tested the effects of each procedure separately using different instruments. The principal objective of this study was to determine and compare the effects of RK and PRK on the quality of vision as measured by CS and GD using several commercially available instruments.PATIENTS AND METHODSSixty consecutive eyes undergoing RK (30 eyes) or PRK (30 eyes) were enrolled in the study. Inclusion criteria for both groups were the same: spherical equivalent of cycloplegic refraction between 1.50 and 6.00 diopters of myopia, astigmatism of 1.25 D or less, the absence of ocular disease, and age 18 years or older. Patients in the RK group were excluded from the study if they had astigmatic keratotomy as an enhancement to their initial surgery. Each excluded patient was replaced by the next eligible consecutive patient so that the final number of eyes in the RK group would be 30. However, enhancement of the radial incisions did not exclude a patient from the study.Detailed ocular history and medical history were obtained and a complete ophthalmological examination, including manifest and cycloplegic refraction, was performed preoperatively. Uncorrected and best spectacle−corrected visual acuity was measured with Early Treatment Diabetic Retinopathy Study (ETDRS) charts. Informed consent was obtained from each patient before enrollment in the study.Surgical procedures were performed with topical anesthesia by 1 of 2 experienced surgeons (R.D.S., K.P.T.). Radial keratotomy consisted of 4 to 8 radial bidirectional incisions with a central clear zone of 3- to 4.5-mm diameter. Photorefractive keratectomy was performed using an excimer laser (Summit Excimer UV 200/OmniMed Excimer Laser, Summit Technology Inc, Waltham, Mass). After mechanically debriding the central epithelium, photoablation was performed using zones of 5 mm (20 eyes) or 6.5 mm (10 eyes), a repetition rate of 10 Hz, and fluence of 180 mJ/cm2.All patients had a complete ophthalmological examination at 1, 3, and 6 months postoperatively. The degree of subepithelial haze after PRK (none, trace, mild, moderate, and severe) was determined by comparison to reference photographs.CS AND GD TESTING METHODSFour devices were used to test CS and GD preoperatively and at the first, third, and sixth postoperative months. All tests were performed with the patient wearing his or her manifest refraction.The CSV 1000 (VectorVision, Dayton, Ohio) consists of a retroilluminated translucent chart with a light level that is automatically adjusted to 85 candelas per square meter (cd/m2). The chart presents vertical sine wave gratings at 4 spatial frequencies: 3, 6, 12, and 19 cycles per degree (cpd). Each row presents 8 pairs of circular patches containing sine waves of a single spatial frequency. For each pair, 1 patch presents a grating and the other patch is blank. Contrast of the gratings decreases from left to right across the row. At a testing distance of 2.4 m (8 ft), the patient was asked to indicate whether the grating appeared in the top patch or the bottom patch for each pair. The contrast level of the last correct response in each row was recorded as the contrast threshold.The MCT (Multivision Contrast Tester) 8000 (Vistech Consultants, Dayton) is a table unit that has 5 slides for CS testing. Each slide has 7 sine wave grating patches arranged in a circle. The contrast of the gratings on each slide decreases from patch 1 to 7 while the spatial frequency remains constant (1.5, 3, 6, 12, or 18 cpd). The gratings randomly vary in their orientation (vertical or tilted to the left or to the right). For CS testing, target illumination was calibrated by the examiner to 130 cd/m2. At a viewing distance of 45 cm, the observer was asked to report the orientation of the lowest contrast patch visible in each slide. This was considered the contrast threshold for that frequency. Glare testing was performed in the same way, but target illumination was decreased to 3.5 cd/m2. A central light of 75 lux was used as a glare source to simulate an automobile headlight.The Pelli-Robson chart (Clement Clark, Columbus, Ohio) is a wall chart that has 8 rows of 6 Sloan letters. The letter size is equivalent to the 20/640 Snellen line. The letters are arranged in groups of 3 (ie, triplets). The contrast decreases from one triplet to the next by steps of about 0.15 log units, ranging from about 100% contrast at the upper left corner to 0.9% contrast at the lower right corner. The acceptable range of chart illumination is between 60 and 20 cd/m2. At 1 m from the chart, the patient was instructed to make a single attempt to name each letter on the chart, starting with the dark letters in the upper left-hand corner. Every letter read correctly adds 0.05 log units to the score.The BAT (Brightness Acuity Tester, Mentor O&O, Santa Barbara, Calif) consists of a 60-mm-diameter hemisphere with a white diffusing surface and a 12-mm central aperture. Using the medium intensity brightness (2500 foot-candles), the patient was instructed to hold the BAT vertically so that the ETDRS chart could be seen through the central aperture and the best-corrected visual acuity was measured.Patients in both groups were asked to respond to a questionnaire about the quality of their vision at the postoperative 3- and 6-month visits. Patients were asked whether their vision was any different at night than during the day and to compare the quality of their vision in the treated eyes with their vision prior to surgery. They were also asked to rate the following measures on a 5-point scale: satisfaction with quality of vision, the severity of glare, problems driving at night, and blurring of vision. In addition, patients were asked if they had problems with glare in 21 different situations.STATISTICAL METHODSAll CS data (with and without glare) were converted to a logarithmic scale and analyzed by 2 methods. In the first method, a paired ttest was used to compare preoperative and postoperative means of CS at each spatial frequency at each postoperative visit, and to compare the mean change in CS at each spatial frequency at each postoperative visit after both procedures. In the second method, the mean percent change in CS was calculated after averaging the percent change in CS from the preoperative value at all spatial frequencies using the following formula: percent change = [postoperative value − preoperative value] × [100/preoperative value]. The mean percent change after both procedures was then compared at each follow-up visit using a ttest. This method has the advantage of producing a single value for CS at each visit, which facilitates comparison of CS measured by multiple devices at multiple spatial frequencies. However, this method did not permit analysis of CS at particular spatial frequencies.Brightness acuity test results were analyzed using a paired ttest to compare (1) the postoperative mean best-corrected visual acuity under the effect of glare at each visit with its preoperative value and (2) the mean change in mean best-corrected visual acuity under the effect of glare after RK with the corresponding value after PRK.The mean percent change in CS after both procedures was correlated with the surgical variables using a correlation coefficient. A ttest was used to compare the mean percent change in CS of eyes with different degrees of haze after PRK. The scores of the questionnaire were compared with the mean change in CS at each spatial frequency and the mean change in visual acuity at the third and sixth postoperative months.RESULTSThe mean (±SD) age of patients was 44.4±7.9 in the RK group and 37.8±8.4 in the PRK group. The mean preoperative manifest refraction in both groups was −3.9±1.4. Nine women were included in both groups, while the number of men was 14 in the RK group and 19 in the PRK group. The number of eyes examined at each follow-up visit is shown in the tabulation below. See table graphicCS RESULTSContrast sensitivity measured with the CSV 1000 was significantly decreased after RK at 3 and 6 cpd at all follow-up visits and at 12 cpd at the third postoperative month (Table 1). Photorefractive keratectomy significantly reduced CS at 6 cpd at all follow-up visits, 12 cpd at the 1- and 6-month visits, and 18 cpd at all visits. The only statistically significant difference between the 2 procedures was at 18 cpd at the first month, where PRK reduced CS more than RK.Table 1. Contrast Sensitivity Results*See table graphicRadial keratotomy did not consistently affect CS as measured with the MCT 8000, which was significantly decreased at 6 cpd at the 6-month visit and significantly increased at 18 cpd at the 1-month visit. Photorefractive keratectomy significantly decreased CS at 1.5 cpd at the first and sixth months, 3 cpd at the first and sixth months, 6 cpd at all visits, 12 cpd at the first month, and 18 cpd at the first month. Photorefractive keratectomy tended to reduce CS early (1 month) and at low and middle spatial frequencies. Photorefractive keratectomy consistently reduced CS more than RK, reaching statistical significance at 1 month at all spatial frequencies.Using the Pelli-Robson chart, CS was significantly decreased at 6 months after RK and at 1 month after PRK. There was no statistically significant difference between the effect of the 2 procedures on CS using this test.GLARE TESTING RESULTSRadial keratotomy consistently reduced CS with glare as measured with the MCT 8000 (Table 2). This effect was significant at the third and sixth postoperative months at all spatial frequencies and was greater at higher spatial frequencies. Photorefractive keratectomy significantly reduced CS with glare at 1 month. The effect seemed to persist with time at lower spatial frequencies, but there was a trend toward recovery of CS at higher spatial frequencies. The 2 procedures were comparable at low spatial frequencies. Photorefractive keratectomy reduced CS with glare more than RK at the first month, while RK reduced it more at the 3- and 6-month visits.Table 2. Glare Disability Results*See table graphicThe BAT showed no significant effect of RK on visual acuity under the effect of glare at any postoperative visit, while PRK caused a statistically significant reduction in visual acuity under the effect of glare at 1 month, with partial recovery at 3 and 6 months. There was no statistically significant difference in the change in visual acuity under the effect of glare after either surgical procedure at any postoperative visit. However, visual acuity with glare was consistently better after RK than after PRK at all visits.QUESTIONNAIREThree months after PRK, a significant negative correlation was found between the mean change in CS at high spatial frequencies and (1) the number of glare situations using the CSV 1000, and (2) the scores of blurring using the MCT 8000. This significant correlation was not found 6 months after PRK. No consistent significant correlation was found between the scores of the questionnaire and (1) the mean change in CS at any spatial frequency using all instruments after RK, and (2) the mean change in visual acuity score using the BAT after both procedures.SURGICAL VARIABLESThere was no statistically significant correlation between percent change in CS and either the number of radial incisions or the diameter of the central clear zone in RK. Also, no statistically significant correlation was found between the percent change in CS and either the number of pulses or the diameter of the ablation zone in PRK.SUBEPITHELIAL HAZE AFTER PRKNo statistically significant correlation was found between the percent change in CS and the severity of subepithelial haze at any visit after PRK. No eyes with mild or moderate haze were observed in the first month. In the third month, eyes with no haze and trace haze were considered one group and eyes with mild and moderate haze were considered another group (due to the small number of eyes with each degree of haze). There were only 18 eyes at the 6-month follow-up visit when the statistical analysis was performed. However, the presence of only 3 eyes with mild or moderate degree of haze at this visit indicates that haze faded with time.COMMENTCS AFTER RK AND PRKIn general, CS decreased after RK and PRK when measured with the 3 devices used in the study. However, the 2 surgical procedures differed with respect to the region of the CS function affected, the time course and magnitude of this reduction in CS, and the device demonstrating this effect (Figure 1). After RK, a significant reduction in CS was demonstrated with the CSV 1000 at the lower frequencies at all the postoperative visits and with the Pelli-Robson chart at the sixth month. The MCT 8000 demonstrated no consistent effect on CS after RK. On the other hand, PRK caused a significant reduction in CS at the middle and high spatial frequencies with the CSV 1000 and the lower and middle spatial frequencies with the MCT 8000 (Table 1).Percent change in contrast sensitivity after radial keratotomy (RK) and photorefractive keratectomy (PRK) using the CSV 1000, the Pelli-Robson chart, and the MCT (Multivision Contrast Tester) 8000.The reduction in CS up to 6 months after RK is similar to the effect reported by previous investigators. Krasnov et alobserved a statistically significant decrease in CS after RK compared with baseline values during the first month after surgery, a minimal difference 2.5 to 4 months after surgery, and no difference 10 to 12 months after surgery. The Prospective Evaluation of Radial Keratotomy (PERK) investigators found an initial, statistically significant decrease in CS in the eyes that were operated on compared with the contralateral eyes that were not operated on. However, this difference disappeared by about 24 months.The PERK investigators considered the average CS differences between the operated- and unoperated-on eyes not to be clinically meaningful (ie, not to affect visual performance), since CS values for the surgically treated and untreated eyes were within the range of previously published reports of normative populations.Tomlinson and Carolinefound that the CS function was significantly reduced 1 year postoperatively in the RK-treated eyes compared with eyes that did not undergo RK. In contrast to the previous results, Olsen and Andersonfound no statistically significant change in CS 1 month after RK using the MCT 8000. They did not report CS data beyond 1 month. Their results are similar to our results obtained with the MCT 8000.Loss of CS after RK may be explained by light scattering from the tips of the radial scars and irregular astigmatism in or near the central clear zone.In time, the radial scars become thinner and less denseand the central clear zone regains its regularity. This may explain the return of CS to its preoperative value after about 1 year.The reduction of CS after PRK in our study is similar to that observed by Ambrosio et al,who found a loss of CS at intermediate spatial frequencies 1 month after PRK. Six months after surgery, however, eyes in their PRK group (which matched the myopic range in our study) showed a full recovery of static CS function but a persistent sensitivity loss for dynamic patterns.In our study, dynamic CS was not measured, and CS was still lower than its preoperative value at 6 months after PRK when measured with all devices. Ficker et alfound reduced CS with the Pelli-Robson chart at 12 months postoperatively. This is similar to our results with the Pelli-Robson chart. However, this reduction was statistically significant only at the first postoperative month in our study. Shimizu et alobserved a reduction in CS with night vision that was still within normal range. Butuner et aldemonstrated a significant reduction in CS in PRK-treated eyes compared with the control group.In contrast to the previous results, Essente et alreported that the mean CS values obtained in their PRK group (which corresponded to the PRK group in our study) were within normal range during all of the follow-up period except for a slight loss of CS at the highest frequencies 2 months after PRK. They also reported that CS returned to baseline levels 3 months after surgery. Eiferman et aldemonstrated no change in CS at the third and sixth postoperative months compared with preoperative values. The small number of eyes in the last 2 studies may explain the difference between their results and ours. Sher et al,using the Pelli-Robson chart and the MCT 8000, found no statistically significant difference between CS before and 3 months after surgery.GD AFTER RK AND PRKNighttime GD (measured with the MCT 8000) was significant after RK at the third and sixth postoperative months at all spatial frequencies. This effect increased with time up to the sixth month and was greater at higher spatial frequencies (Table 2). The latter observation is difficult to explain because the scars become less dense by the sixth month. The statistically nonsignificant decrease in CS under the effect of a central glare source at the first postoperative month correlates well with the results of Olsen and Anderson,who found that, using the MCT 8000 with the central glare source on, RK did not significantly reduce CS function 1 month after surgery. No daytime GD was detected after RK using the BAT. This is consistent with results of the PERK Study, in which no effect of glare on visual acuity was found 1 year after RK.After PRK, significant nighttime GD was demonstrated with the MCT 8000 at the first postoperative month at all spatial frequencies. This effect seems to persist with time at lower spatial frequencies, but there was a trend toward recovery of CS at higher spatial frequencies (Table 2). We hypothesize that an irregular corneal surface causes early postoperative glare after PRK. The BAT demonstrated significant daytime GD only at the first month after PRK. These results are different from those of Ambrosio et aland Eiferman et al,who found, using the BAT, that glare did not affect visual acuity up to 6 months after PRK. Seiler et aldemonstrated a loss in glare vision that correlated significantly with the amount of attempted correction, with a greater loss of glare vision following higher corrections. We did not correlate the loss in visual acuity with glare with the attempted correction, as dividing the limited number of eyes with a narrow range of myopia (−2.00 to −4.00 D) would not have permitted meaningful statistical analysis.COMPARISON BETWEEN THE EFFECT OF RK AND PRK ON CSDifferent results were obtained with the different devices used in this study to compare the effects of RK and PRK on CS. No significant difference was found with the CSV 1000 and the Pelli-Robson charts. The MCT 8000, on the other hand, demonstrated that PRK significantly decreased CS more than RK at all spatial frequencies at the first postoperative month only. However, this difference disappeared by the third month.COMPARISON BETWEEN THE EFFECTS OF RK AND PRK ON GDThe BAT demonstrated no significant difference between the 2 procedures with respect to GD up to 6 months postoperatively. The MCT 8000 demonstrated that PRK caused significantly more nighttime GD than RK only at the first postoperative month.CLINICAL SIGNIFICANCE OF CS AND GD TESTING RESULTSThe absence of a strong correlation between the results of the questionnaire and the objective CS and GD scores may allow us to conclude that any postoperative decrease in CS or increase inGD does not affect the everyday visual experience of patients. In the PERK Study, as well, no correlation was found between the CS function measured under photopic conditions and the glare index from a psychometric questionnaire 1 year after RK.All postoperative mean values of CS (with and without glare) and visual acuity under the effect of glare fell between the corresponding preoperative 5th and 95th percentile values for the same patient populations. This was true for all the spatial frequencies at all postoperative visits. These data further support the conclusion that the statistically significant reduction of CS or increase in GD after RK and PRK may not be clinically significant.The different results obtained by the devices used to measure CD illustrate the importance of standardization of CS testing parameters (eg, target type and illumination, testing distance, methods of reaching contrast threshold). Such standardization will make it more meaningful to compare the effect of different surgical procedures and different disease processes on CS. Also, it will be possible to measure the CS of normative populations and use this information to help determine the clinical relevance of CS results.Our understanding of CS and GD after RK and PRK can be further extended by including more patients, following them up for longer periods of time, studying the role played by surrounding lighting conditions, pupillary diameter, and postoperative magnification on CS and GD, including dynamic CD testing, and correlating CS and GD scores after refractive procedures and corneal topography.DJNadlerGlare and contrast sensitivity in cataracts and pseudophakia.In: Nadler MP, Miller D, Nadler DJ, eds. Glare and Contrast Sensitivity for Clinicians.New York, NY: Springer-Verlag; 1990:53-65.DDKochGlare and contrast sensitivity for the clinician.J Cataract Refract Surg.1989;15:158-164.LFJindraVZemonContrast sensitivity testing: a more complete assessment of vision.J Cataract Refract Surg.1989;15:141-148.APGinsburgThe evaluation of contact lenses and refractive surgery using contrast sensitivity.In: Dabezie OH, ed. Contact Lenses: The CLAO Guide to Basic Science and Clinical Practice.New York, NY: Grune & Stratton; 1987;56:1-19.JWolfeAn introduction to contrast sensitivity testing.Nadler MP, Miller D, Nadler DJ, eds. Glare and Contrast Sensitivity for Clinicians.New York, NY: Springer-Verlag; 1990;2:3-23.DMillerOptics and refraction: a user-friendly guide.In: Podos SM, Yanoff M, eds. Textbook of Ophthalmology.London, England: CV Mosby; 1994;1(7):14-24.SMasketGlare disability and contrast sensitivity function in the evaluation of symptomatic cataract.Ophthalmol Clin North Am.1991;4:365-379.NSJaffeGlare and contrast: indications for cataract surgery.J Cataract Refract Surg.1986;12:372-375.MAbrahammsonJSjostrandImpairment of contrast sensitivity function as a measure of disability glare.Invest Ophthalmol Vis Sci.1986;27:1131-1136.MMKrasnovSEAvetisovNVMakashovaVRMamikonianThe effect of radial keratotomy on contrast sensitivity.Am J Ophthalmol.1988;105:651-654.APGinsburgGOWaring IIIEBSteinbergContrast sensitivity under photopic conditions in the Prospective Evaluation of Radial Keratotomy (PERK) Study.Refract Corneal Surg.1990;6:82-91.ATomlinsonPCarolineEffect of radial keratotomy on the contrast sensitivity function.Am J Optom Physiol Opt.1988;65:803-808.HOlsenJAndersenContrast sensitivity in radial keratotomy.Acta Ophthalmol (Copenh).1991;69:654-658.LRTrickJHartsteinInvestigation of contrast sensitivity following radial keratotomy.Ann Ophthalmol.1987;19:251-254.APGinsburgDWEvansMWCannon JrCOwsleyPMulvannyLarge-sample norms for contrast sensitivity.Am J Optom Physiol Opt.1984;61:80-84.GMLongDLPennNormative contrast sensitivity function: the problem of comparison.Am J Optom Physiol Opt.1987;64:131-135.GOWaringEBSteinbergLAWilsonSlit-lamp microscopic appearance after corneal wound healing.Am J Ophthalmol.1985;100:218-224.GAmbrosioGCennamoRDMarcoLLoffredoNRosaASebastianiVisual function before and after photorefractive keratectomy for myopia.J Refract Corneal Surg.1994;10:129-136.LAFickerAKBatesADMcGSteeleExcimer laser photorefractive keratectomy for myopia: 12 month follow-up.Eye.1993;7:617-624.KShimizuSAmanoSTanakaPhotorefractive keratectomy for myopia: one-year follow-up in 97 eyes.J Refract Corneal Surg.1994;10(suppl):S178-S187.ZButunerDBElliotHVGimbelSSlimmonVisual function one year after excimer laser photorefractive keratectomy.J Refract Corneal Surg.1994;10:625-630.SEssenteNPassarelliLFalcoFPassaniDGuidiContrast sensitivity under photopic conditions in photorefractive keratectomy: a preliminary study.J Refract Corneal Surg.1993;9(suppl):S70-S72.RAEifermanKPO'NeillDRForgeyYDCookExcimer laser photorefractive keratectomy for myopia: six-month results.Refract Corneal Surg.1991;7:344-347.NASherVChenRABowersThe use of the 193-nm excimer laser for myopic photorefractive keratectomy in sighted eyes: a multicenter study.Arch Ophthalmol.1991;109:1525-1530.TSeilerFHolschbachDMatthiasBJeanUGenthComplications of myopic photorefractive keratectomy with the excimer laser.Ophthalmology.1994;101:153-160.CSCartwrightLBBourqueMLynnGOWaring IIIThe PERK Study GroupRelationship of glare to uncorrected visual acuity and cycloplegic refraction 1 year after radial keratotomy in the Prospective Evaluation of Radial Keratotomy (PERK) Study.J Am Optom Assoc.1988;59:36-39.Accepted for publication July 25, 1997.Reprints: R. Doyle Stulting, MD, PhD, Department of Ophthalmology, Emory Eye Center, 1365-B Clifton Rd NE, Atlanta, GA 30322 (e-mail: [email protected]).

Larsson, Lill-Inger; Alm, Albert

1998 JAMA Ophthalmology

doi: 10.1001/archopht.116.1.19pmid: 9445204

ObjectiveTo measure the effect of topically applied 2% dorzolamide hydrochloride (Trusopt, Merck & Co Inc, Whitehouse Station, NJ) and different doses of orally administered acetazolamide (Diamox, Lederle Ophthalmic Pharmaceuticals, Pearl River, NY), alone and in combination, on aqueous humor flow.DesignA randomized, double-masked, placebo-controlled study of 20 human subjects was carried out. Aqueous humor flow was measured by clearance of topically applied fluorescein. Serum standard bicarbonate and serum acetazolamide levels were analyzed.ResultsTreatment with dorzolamide reduced aqueous flow by 17%, and a maximum dose of acetazolamide alone reduced flow by 29%. Increasing doses of acetazolamide alone gradually decreased flow, while small doses of acetazolamide did not suppress flow further when dorzolamide was already applied topically. Serum acetazolamide concentrations rose with increasing doses of acetazolamide. Serum standard bicarbonate levels were all in the normal range.ConclusionsTreatment with dorzolamide reduced aqueous humor flow statistically significantly (2.50 µL/min vs 3.00 µL/min; P=.001) compared with placebo, but less than a maximum dose of acetazolamide. Small doses of acetazolamide added to dorzolamide treatment did not further enhance the decrease in flow. Since there was no metabolic acidosis as measured by plasma levels of standard bicarbonate, the decrease in aqueous flow could be attributed to the direct action of the carbonic anhydrase inhibitors on the carbonic anhydrase enzymes. It is concluded that the smaller effect of dorzolamide, as compared with acetazolamide, is due to insufficient inhibition of at least 1 of the 2 carbonic anhydrase isozymes involved in aqueous humor production.AN EXCESS of bicarbonate in the aqueous humorand the presence of carbonic anhydrase (CA) activity in the ciliary body of the rabbitwere reported in the 1950s, suggesting that inhibition of the enzyme might lead to a decrease in aqueous formation. Shortly afterwards, the inhibitor acetazolamide was introduced for the treatment of glaucoma.Systemic administration of CA inhibitors reduces intraocular pressure by suppressing aqueous humor formation.However, the use of these compounds in the routine medical management of glaucoma has been limited owing to adverse effects.Topical use of CA inhibitors was thought of as a solution to avoid systemic complications. Continuous delivery of acetazolamide soaked in a soft contact lens reduced intraocular pressure,but administration of acetazolamide as eye drops or as a subconjunctival injection had little effect.The explanation for this could be that the concentration of the CA inhibitor in the ciliary body was too low to suppress flow in the latter experiment, while it was high enough in the former study. Maren has shown that approximately 99% of the enzymatic activity needs to be inhibited to result in reduced aqueous humor production. Intensive research to develop a topically effective analog to acetazolamide has identified several promising new agents. Maren and colleaguesworked with several water-soluble compounds that also could penetrate the limiting layers of the eye, and trifluoromethazolamidewas found to be an interesting substance. Because of its instability, it was never tested in humans. Ponticello and coworkersdiscovered a novel class of water-soluble CA inhibitors, the thienothiopyran-2-sulfonamides. Dorzolamide hydrochloride was one of the most promising agents in this group. It was extensively tested clinicallyand was found to lower intraocular pressure satisfactorily.Wang and colleagueshave shown that dorzolamide suppresses aqueous humor flow in monkeys. Yamazaki et alfound increased flare, measured by laser-flare cell photometry, in 6 human subjects given single doses of dorzolamide, and they interpreted the results as evidence for reduced aqueous humor flow. Another topical CA inhibitor, 6-amino-2-benzothiazole-sulfonamide, reduced aqueous humor production in human eyes.A recent study by Maus and coworkerscompared the efficacy of topically administered dorzolamide as a suppressor of aqueous humor flow in humans to that of a maximal oral dose of acetazolamide. Dorzolamide suppressed aqueous humor flow by 17%, while acetazolamide suppressed the flow by 30%. There was no obvious explanation for the lower effect of dorzolamide, which in vitro is an effective inhibitor of CA isozyme II.One possible explanation is that part of the flow reduction obtained with a full dose of acetazolamide is unrelated to local CA inhibition. Acetazolamide causes a metabolic acidosis with time, and it has been suggested that a local acidic environment contributes to the flow reduction.In the previous study by Maus et al,levels of serum bicarbonate were not determined, but the treatment period was short and a marked metabolic acidosis is an unlikely explanation for the difference in potency between the 2 studied drugs. Another explanation is that dorzolamide causes a less complete inhibition of the CAs involved compared with acetazolamide. Recent studies have shown that CA isozyme IV is also involved in the production of aqueous humor.We decided to test these 2 possibilities by determining the effect of dorzolamide and various doses of acetazolamide, alone and in combination, on aqueous humor flow and serum bicarbonate levels.SUBJECTS, MATERIALS, AND METHODSTwenty normal subjects were included in the study. There were 11 women and 9 men and the mean age was 31.7 years (range, 20-49 years). Medical and ophthalmological histories were taken for all subjects. They also underwent an ophthalmic screening examination consisting of visual acuity testing, slitlamp examination, applanation tonometry, and ophthalmoscopy. Exclusion criteria were ocular disease, systemic disease requiring long-term medical treatment, inability to comply with tonometry or fluorophotometry, an intraocular pressure difference between the 2 eyes greater than 3 mm Hg, history of kidney stones, and known drug hypersensitivity (especially to sulfonamide derivatives). The research protocol was approved by the Ethical Committee of Uppsala University, Uppsala, Sweden, and informed consent was obtained from all participants.The study was performed in 4 parts and the sequence of the 4 parts was randomized. In part 1, the effect of 2% dorzolamide vs placebo was studied when the subjects received oral administration of placebo capsules. Parts 2 through 4 were identical to part 1 except that the oral placebo capsules were replaced by acetazolamide capsules; 31.3 mg in part 2, 62.5 mg in part 3, and 250 mg in part 4. There was a washout period of at least 14 days between all parts of the study to ensure complete elimination of the drugs.The study was randomized, double-masked, and placebo-controlled. The dorzolamide and placebo eye drops, as well as the acetazolamide and placebo capsules, were given by random assignment and were administered from identically appearing containers labeled by subject number, sequence, and, where appropriate, eye. The active ingredient in the eye drops was 2% dorzolamide (Trusopt, Merck & Co Inc, Whitehouse Station, NJ), and artificial tears were used as placebo eye drops (Isopto-Plain, Alcon Laboratories, Ft Worth, Tex). Acetazolamide tablets (Diamox, Lederle Ophthalmic Pharmaceuticals, Pearl River, NY), ground to a fine powder, were used as the active drug in the oral capsules, and identically appearing placebo capsules were prepared without the active ingredient. Half the subjects received dorzolamide in the right eye, and half received it in the left eye. Also, the oral capsules were equally randomized between the different parts of the study.Each part of the study was performed on 2 consecutive days (Figure 1). On day 1 the subjects reported to the test area at 8 AM. One drop (&ap;20 µL) of 2% dorzolamide hydrochloride was given in one eye, and 1 drop of placebo in the other eye. This procedure was repeated at noon and 5 PM. The subjects were instructed to awaken at 2 AM on day 2 and instill 1 drop of 2% fluorescein into each eye 3 to 5 times, according to age, at 5-minute intervals and then return to sleep. The subjects then reported to the research area at 8 AM and fluorophotometric measurements of the cornea and anterior chamber were performed. Fluorophotometry was repeated every other hour until 4 PM. After the last fluorophotometric reading at 4 PM, the intraocular pressure was measured with a Goldmann tonometer. On day 2, the same eye drops were given after the measurements at 8 AM and at noon. In addition to the eye drops, an oral capsule of acetazolamide or placebo was given at 8 AM and noon. After the tonometry at 4 PM, 2 blood samples were drawn for analysis of acetazolamide and standard bicarbonate levels in serum.Figure 1.Sequence of events in experimental protocol.Fluorescence was measured with a fluorophotometer (Fluorotron Master, Coherent Radiation, Palo Alto, Calif). Aqueous humor flow was calculated from the clearance of fluorescein at each 2-hour interval with the following equation:Clearance = ΔM/(Ca× Δt),where ΔMis the loss of mass of fluorescein in the combined cornea and anterior chamber during an interval (Δt), and Cais the average concentration in the anterior chamber during the interval, estimated from the initial and final fluorescence and assuming a single exponential decay. Aqueous humor flow was determined from the rate of clearance of fluorescein after subtracting the presumed rate of diffusional clearance (0.25 µL/min).The blood samples that were drawn on day 2 were handled immediately. Analysis of serum standard bicarbonate levels was performed at the chemical laboratory at Uppsala University Hospital, Uppsala Sweden. The reference range for serum bicarbonate was 23 to 33 mmol/L. The samples for acetazolamide level analysis were centrifuged, and the plasma was collected and frozen at 70°C. At the end of the study all these frozen samples were sent to David Berry, PhD, Medical Toxicology Unit, Guy's & St Thomas' Hospital Trust, London, England, for analysis of acetazolamide levels in serum.The Student 2-sided ttest for paired samples was used for the statistical analysis. A P<.05 was considered statistically significant. In previous studies, the normal aqueous humor flow rate in daytime has been measured to be 2.75±0.63 µL/min (mean±SD).A sample size of 20 in each group would provide a power of 95% for detecting a true difference of 20% between the 2 eyes.RESULTSOne subject had marked myopia (−3.5 diopters) in both eyes in the evening of day 2 of her second part of the study. The myopia slowly resolved and the refraction returned to normal within 48 hours. The randomization code was broken for this subject, and it was revealed that she had received the highest dose of acetazolamide (250 mg given twice) on this particular day. She was thereafter withdrawn from the study, but data from the 2 already completed parts were included in the analysis. During the course of the study 1 female subject was diagnosed with a malignant breast tumor and therefore was excluded. The remaining 18 subjects completed the study.The results on the aqueous humor flow in eyes treated with placebo eye drops and corresponding results for dorzolamide-treated eyes are presented in Table 1. The lowest dose of acetazolamide had no statistically significant effect but higher doses of acetazolamide gradually suppressed aqueous humor flow in eyes receiving topical placebo. The maximum dose of 250 mg of acetazolamide given twice (corresponding to a total daily dose of 1000 mg of acetazolamide) reduced flow by 29% compared with oral placebo capsules (P<.001). Dorzolamide alone suppressed flow by 17% compared with placebo (2.50 µL/min vs 3.00 µL/min; P=.001), which was almost twice the flow suppression obtained with an oral dose of acetazolamide of 62.5 mg given twice (corresponding to a daily dose of 250 mg of acetazolamide), but less than that of an acetazolamide dose of 250 mg given twice (Table 1). In the dorzolamide-treated eyes, the lower doses of acetazolamide did not add any further suppression of flow; it was only the highest dose of acetazolamide (250 mg given twice) that reduced the aqueous humor flow in a statistically significant way (16% compared with oral placebo capsules [P<.001]).Table 1. Aqueous Humor Flow (8 AM-4 PM) in Eyes Receiving Topical Placebo and Eyes Treated With Topical 2% Dorzolamide*See table graphicWhen equal doses of acetazolamide were given, there was a statistically significant difference in flow between eyes receiving placebo and eyes receiving dorzolamide at the lower doses of acetazolamide, but not when the maximum dose was administered (Figure 2). When untreated eyes (placebo drops and placebo capsules) were compared with maximally treated eyes (dorzolamide drops and 250-mg acetazolamide capsules), there was a difference in flow of 30% (P<.001). The time courses of flow under the different experimental conditions are shown in Figure 3and show the effect of time of day. There was no great difference in reduction of aqueous humor flow between the 2-hour intervals 10 AM to noon and 2 to 4 PM.Figure 2.Aqueous humor flow in untreated (placebo) and dorzolamide-treated eyes when oral capsules of different doses of acetazolamide or placebo are administered. Mean and standard deviations are shown and Pvalues are given.Figure 3.Top, Time course of aqueous humor flow for different doses of acetazolamide in eyes receiving topical placebo. Bottom, Time course of aqueous humor flow for different doses of acetazolamide in eyes treated with topical 2% dorzolamide hydrochloride.The intraocular pressures at 4 PM for eyes receiving placebo and for dorzolamide-treated eyes are summarized in Table 2. The results are in concordance with the results from the flow measurements, but the percent reduction of the intraocular pressure was smaller than the percent reduction of aqueous flow. This difference is expected since the outflow pressure (intraocular pressure minus episcleral venous pressure) rather than the intraocular pressure is reduced in proportion to aqueous humor flow reduction. Also, the subjects all had low initial intraocular pressures.Table 2. Intraocular Pressure (IOP) at 4 PM in Eyes Receiving Topical Placebo and Eyes Treated With Topical 2% Dorzolamide*See table graphicSerum concentrations of acetazolamide at 4 PM on day 2 of each part of the study are given in Table 3, as well as the serum standard bicarbonate levels. The concentration of acetazolamide in serum was increased with higher doses of oral acetazolamide. Serum standard bicarbonate levels were all in the normal range. The suppression of aqueous humor flow increased with rising serum levels of acetazolamide (Figure 4).Table 3. Levels of Serum Acetazolamide and Serum Standard Bicarbonate With Different Doses of Acetazolamide*See table graphicFigure 4.Aqueous humor flow in eyes receiving topical placebo and their corresponding serum levels of acetazolamide at 4 PM, when oral capsules are given.COMMENTThe data from the present study confirm previous results that 2% dorzolamide hydrochloride suppresses aqueous humor formation, but not to the same extent as a maximum dose of acetazolamide.Treatment with dorzolamide alone reduced aqueous flow by 17% in this study, and a maximum dose of acetazolamide alone reduced flow by 29%. Corresponding figures for comparable doses were 17% and 30% in the previously mentioned study.Increasing doses of acetazolamide gradually decreased the aqueous flow. However, small doses of orally administered acetazolamide did not suppress flow further when dorzolamide was already applied topically. Only the highest dose of acetazolamide (250 mg) resulted in accentuated suppression of the aqueous humor flow.In the present study we administered 3 doses of acetazolamide, apart from placebo. The highest dose, 250 mg of acetazolamide given twice (corresponding to a daily dose of 1000 mg of acetazolamide), was chosen to ensure a maximal effect, and also to use the same oral dose as was used in the study by Maus et alto be able to make adequate comparisons of effects on flow. The aim of the study was to see if a low dose of acetazolamide, with few systemic adverse effects, could enhance the aqueous flow suppression obtained with dorzolamide. For that purpose we decided to test 2 low doses of acetazolamide, 31.3 mg and 62.5 mg, and follow the effect on aqueous flow after a first and second administration of these doses. Acetazolamide was administered in gelatin capsules that we expected to be rapidly dissolved in the stomach and not to retard uptake of acetazolamide into plasma. Peak plasma levels after oral acetazolamide are reached about 2 hours after the dose and start to decline rapidly after 6 hours.Thus, aqueous flow between 10 AM and noon corresponds to the peak effect of the single dose. The effects observed between noon and 2 PM and between 2 and 4 PM correspond to a dose that is somewhat higher than the single dose, because another dose of acetazolamide was given at noon. Neither dose caused a marked improvement of the effect on aqueous flow after noon, which indicates that all 3 doses were at flat parts of the dose-response curve, the 2 lower doses at the low end and the highest dose at the top.The reduction of aqueous humor flow by dorzolamide treatment alone was almost twice that obtained with an oral dose of 62.5 mg of acetazolamide given twice, but definitely lower than the reduction in flow obtained by 250 mg of acetazolamide given twice. The addition of 31.3 or 62.5 mg of acetazolamide to dorzolamide did not suppress the aqueous humor production any further (P=.22 and P=.23). If the mechanism of action is identical for the 2 drugs, some additivity would be expected, at least with 62.5 mg, which caused a statistically significant effect in eyes that received the topical placebo. The maximal intraocular pressure–lowering effect has earlier been shown to occur at plasma concentrations of acetazolamide between 5 and 10 µg/mL.The mean plasma concentration achieved with 62.5 mg of acetazolamide given twice, 4.3 µg/mL (Table 3), was just below this level.One explanation for the lack of additivity could be that dorzolamide and acetazolamide act differently on the different CA isozymes. There are at least 7 different CA isozymes, and 2 of them have been found in the ciliary epithelium; the cytoplasmic CA IIand the membrane-bound CA IV.Both CA II and CA IV seem to coexist in epithelial cells that perform acid-base work, ie, in cells secreting hydrogen ions either at the luminal or basolateral membranes.It has been suggested that CA IV is the critical enzyme for secretion,because many secretory cells have vectorial properties, with the secretion of ions directed toward basal or apical surfaces. In a recent study by Matsui and coworkers,both CA isozymes in the ciliary epithelium were suggested to be involved in aqueous humor production: the cytoplasmic (CA II) and the membranal (CA IV). The action of the nonpigmented epithelium basolateral membranal CA IV was suggested to be linked to the chloride-bicarbonate exchanger. The inhibitory concentration of dorzolamide for human CA II was 0.2 nmol/L.Marenhas reported that the equilibrium dissociation constants at room temperature for dorzolamide with CA II and CA IV are 8 and 300 nmol/L, respectively, and that with an expected tissue concentration of 10 µmol/L after application of a 2% dorzolamide solution, the fractional inhibitions of CA II and CA IV are 0.999 and 0.970, respectively.If the effect of the 2 CA inhibitors is explained solely by enzyme inhibition, our results indicate that a part of the active enzyme is not affected by dorzolamide and that this portion of CA is affected only when acetazolamide is administered in a dose large enough to be effective on its own. It seems reasonable to assume that dorzolamide effectively inhibits only 1 of the isozymes involved in aqueous humor production. Whether that is due to the higher equilibrium dissociation constant for one of them (CA IV) or to the inability or difficulty of topically applied CA inhibitors to reach and maintain an effective inhibitory concentration at the membrane or in the cytosol cannot be detertmined from the present study.Another possibility is that acetazolamide has an effect on aqueous flow that is not related to inhibition of CA in the ciliary processes. Systemic administration of these agents can cause systemic electrolyte disturbances, primarily systemic acidosis, which has been suggested to contribute to the ocular effects.However, we did not find any metabolic acidosis as measured by plasma levels of standard bicarbonate, which makes this explanation less likely.Addition of twice-daily 2% dorzolamide to the regimen of patients with glaucoma already receiving treatment with timolol or betaxolol has produced a clinically beneficial increase in ocular hypotensive effects.In the present study, we found that treatment with dorzolamide reduced aqueous humor flow and thereby intraocular pressure, but to a lower extent than a maximum dose of orally administered acetazolamide. Small doses of acetazolamide added to dorzolamide treatment did not further enhance the decrease in flow and intraocular pressure. Dorzolamide has the advantage over high doses of acetazolamide that it is well accepted by patients, because intolerable systemic effects are much less likely to occur. However, further studies need to be pursued to know what kind of effects different combinations of dorzolamide and other aqueous suppressors might have on intraocular pressure and aqueous humor flow.VEKinseyComparative chemistry of aqueous humor in posterior and anterior chambers of rabbit eye.Arch Ophthalmol.1953;50:401-417.PWistrandCarbonic anhydrase in the anterior uvea of the rabbit.Acta Physiol Scand.1951;24:144-148.BBeckerDecrease in intraocular pressure in man by a carbonic anhydrase inhibitor, Diamox: a preliminary report.Am J Ophthalmol.1954;37:13-15.WMGrantRRTrotterDiamox (acetazolamide) in the treatment of glaucoma.Arch Ophthalmol.1954;51:735-739.GMBreininHGörtzCarbonic anhydrase inhibitor acetazolamide (Diamox): a new approach to the therapy of glaucoma.Arch Ophthalmol.1954;52:333-348.PRLichterLPNewmanNCWheelerOVBeallPatient tolerance to carbonic anhydrase inhibitors.Am J Ophthalmol.1978;85:495-502.ZFriedmanRCAllenSMRaphTopical acetazolamide and methazolamide delivered by contact lenses.Arch Ophthalmol.1985;103:963-966.RHFossLocal application of Diamox: an experimental study of its effect on the intraocular pressure.Am J Ophthalmol.1955;39:336-339.THMarenCarbonic anhydrase: chemistry, physiology, and inhibition.Physiol Rev.1967;47:595-781.THMarenLJankowskaGSanyalHFEdelhauserThe transcorneal permeability of sulfonamide carbonic anhydrase inhibitors and their effect on aqueous humor secretion.Exp Eye Res.1983;36:457-480.ASteinRPinkeTKrupinThe effect of topically administered carbonic anhydrase inhibitors on aqueous humor dynamics in rabbits.Am J Ophthalmol.1983;95:222-228.GSPonticelloMBFreedmanCNHabeckerThienothiopyran-2-sulfonamides: a novel class of water-soluble carbonic anhydrase inhibitors.J Med Chem.1987;30:591-597.YKitazawaIAzumaKIwataDorzolamide, a topical carbonic anhydrase inhibitor: a two-week dose-response study in patients with glaucoma or ocular hypertension.J Glaucoma.1994;3:275-279.EALippaL-ECarlsonBEhingerDose response and duration of action of dorzolamide, a topical carbonic anhydrase inhibitor.Arch Ophthalmol.1992;110:495-499.EALippaJSSchumanEJHigginbothamMK-507 versus sezolamide: comparative efficacy of two topically active carbonic anhydrase inhibitors.Ophthalmology.1991;98:308-313.FPGunningELGreveAMBronTwo topical carbonic anhydrase inhibitors sezolamide and dorzolamide in Gelrite vehicle: a multiple-dose efficacy study.Graefes Arch Clin Exp Ophthalmol.1993;231:384-388.MWilkersonMCyrlinEALippaFour-week safety and efficacy study of dorzolamide, a novel, active topical carbonic anhydrase inhibitor.Arch Ophthalmol.1993;111:1343-1350.YYamazakiSMiyamotoMSawaEffect of MK-507 on aqueous humor dynamics in normal human eyes.Jpn J Ophthalmol.1994;38:92-96.The MK-507 Clinical Study GroupLong-term glaucoma treatment with MK-507, dorzolamide, a topical carbonic anhydrase inhibitor.J Glaucoma.1995;4:6-10.EStrahlmanRTippingRVogeland the International Dorzolamide Study GroupA double-masked, randomized 1-year study comparing dorzolamide (Trusopt), timolol, and betaxolol.Arch Ophthalmol.1995;113:1009-1016.R-FWangJBSerleSMPodosMFSugrueMK-507 (L-671,152), a topically active carbonic anhydrase inhibitor, reduces aqueous humor production in monkeys.Arch Ophthalmol.1991;109:1297-1299.PHKalinaDJShetlarRALewisLJKullerstrandRFBrubaker6-amino-2-benzothiazole-sulfonamide: the effect of a topical carbonic anhydrase inhibitor on aqueous humor formation in the normal human eye.Ophthalmology.1988;95:772-777.TLMausL-ILarssonJWMcLarenRFBrubakerComparison of dorzolamide and acetazolamide as suppressors of aqueous humor flow in humans.Arch Ophthalmol.1997;115:45-49.JJBaldwinGSPonticelloPSAndersonThienothiopyran-2-sulfonamides: novel topically active carbonic anhydrase inhibitors for the treatment of glaucoma.J Med Chem.1989;32:2510-2513.BBeckerCarbonic anhydrase and the formation of aqueous humor: the Friedenwald Memorial Lecture.Am J Ophthalmol.1955;47:342-361.HMatsuiMMurakamiGCWynnsMembrane carbonic anhydrase (IV) and ciliary epithelium carbonic anhydrase activity is present in the basolateral membranes of the non-pigmented ciliary epithelium of rabbit eyes.Exp Eye Res.1996;62:409-417.RFBrubakerMeasurement of aqueous flow by fluorophotometry.In: Ritch R, Shields MB, Krupin T, eds. The Glaucomas.St Louis, Mo; CV Mosby Co: 1989;1:337-344.CFagerlundPHartvigPLindstromExtractive alkylation of sulphonamide diuretics and their determination by electron-capture gas chormatography.J Chromatogr.1979;168:107-116.DJBerryThe determination of acetazolamide in human plasma by reversed phase HPLC.12th International Symposium on Column Liquid Chromatography, 1988.Abstract # TU-P-282.RFBrubakerFlow of aqueous humor in humans: the Friedenwald Lecture.Invest Ophthalmol Vis Sci.1991;32:3145-3166.WJDixonJFMassey JrIntroduction to Statistical Analysis.New York, NY: McGraw-Hill Book Co; 1969:516.RJDerickGlaucoma therapy: carbonic anhydrase inhibitors.In: Mauger TF, Craig EL, eds. Havener's Ocular Pharmacology.6th ed. St Louis, Mo: Mosby-Year Book Inc; 1994:172-200.AAlmLBerggrenPHartvigMRoosdorpMonitoring acetazolamide treatment.Acta Ophthalmol (Copenh).1982;60:24-34.PJWistrandLCGargEvidence of a high-activity C type of carbonic anhydrase in human ciliary processes.Invest Ophthalmol Vis Sci.1979;18:802-806.YRidderstralePJWistrandWFBrechueMembrane-associated CA activity in the eye of the CA II-deficient mouse.Invest Ophthalmol Vis Sci.1994;35:2577-2584.GLönnerholmPJWistrandMembrane-bound carbonic anhydrase CA IV in the human kidney.Acta Physiol Scand.1991;141:231-234.THMarenCurrent status of membrane-bound carbonic anhydrase.Ann N Y Acad Sci.1980;341:246-258.WFBrechueEKinne-SaffranRKHKinneTHMarenLocalization and activity of renal carbonic anhydrase (CA) in CA-II deficient mice.Biochim Biophys Acta.1991;1066:201-207.THMarenBasic sciences in clinical glaucoma: the development of topical carbonic anhydrase inhibitors.J Glaucoma.1995;4:49-62.MELanghamPMLeeAction of Diamox and ammonium chloride on formation of aqueous humor.Br J Ophthalmol.1957;41:65-92.TKrupinCJOestrickJBassSMPodosBBeckerAcidosis, alkalosis, and aqueous humor dynamics in rabbits.Invest Ophthalmol Vis Sci.1977;16:997-1001.Accepted for publication September 16, 1997.Reprints: Lill-Inger Larsson, MD, PhD, Department of Ophthalmology, Uppsala University Hospital, S-75185, Uppsala, Sweden.

Kruger, Andreas; Matulla, Bettina; Wolzt, Michael; Pieh, Stephan; Strenn, Karin; Findl, Oliver; Eichler, Hans-Georg; Schmetterer, Leopold

1998 JAMA Ophthalmology

doi: 10.1001/archopht.116.1.27

ObjectiveTo study the ocular hemodynamic effects of a 3-month oral treatment with pentoxifylline in patients with nonexudative age-related macular degeneration.DesignDouble-blind, placebo-controlled, randomized, parallel group study.SettingOutpatient clinic of the Department of Ophthalmology, Vienna University, Vienna, Austria, that specializes in age-related macular degeneration.MethodsForty patients with age-related macular degeneration received pentoxifylline (400 mg 3 times a day orally, n=20) or placebo (n=20) for 3 months. Retinal blood flow was assessed by scanning laser Doppler flowmetry and pulsatile choroidal blood flow was assessed by laser interferometric measurement of fundus pulsation amplitude.Main Outcome MeasuresChanges in retinal blood flow and fundus pulsation amplitude.ResultsFour patients receiving pentoxifylline and 3 patients receiving placebo discontinued medication because of nausea. In the remaining subjects, the use of pentoxifylline increased ocular fundus pulsation amplitude (P<.001 vs placebo and baseline). The maximum increase was 28% after 3 months. In contrast, retinal blood flow was not changed by the use of pentoxifylline.ConclusionsA 3-month course of oral pentoxifylline treatment increases choroidal but not retinal blood flow in patients with age-related macular degeneration. These data strongly support the concept that pentoxifylline might be useful in the treatment of age-related macular degeneration. Long-term clinical outcome trials are now warranted to test this hypothesis.ALTHOUGH age-related macular degeneration (AMD) is the most common cause of blindness in Western countries, the pathogenesis of the disease is less well understood.To date, the only treatment of this disease is the application of laser photocoagulation, which is effective for only a few patients. Even in successfully treated eyes, the development of central visual impairment often cannot be prevented. For patients with nonneovascular AMD, there exists no proven prophylactic treatment.Recent studies indicate that blood flow in the choroid is impaired in patients with AMD. A prolonged choroidal filling phase demonstrated by fluorescein angiography has been ascribed to thickening of the Bruch membrane.The use of color Doppler imaging has shown increased pulsatility indexes in ocular vessels, which argues for an increased vascular resistance.Pentoxifylline is a synthetic xanthine derivative, the use of which has been proposed for the treatment of several eye diseases. The possible therapeutic value of the drug is based mainly on the increase in ocular blood flow that has been observed in healthy subjects,in patients with diabetes mellitus,and in patients with branch vein or central retinal vein occlusion.The ability of pentoxifylline to increase blood flow results from its direct vasodilator actionand improved deformability of erythrocytesand leukocytes.Based on these findings, we hypothesized that pentoxifylline treatment may improve ocular blood flow in patients with AMD. We therefore studied its short-term effects over 3 months in a placebo-controlled, double-blind trial. Drug-induced changes in choroidal circulation were assessed with laser interferometric measurement of fundus pulsationand the effect on retinal blood flow was assessed with scanning laser Doppler flowmetry.SUBJECTS AND METHODSSUBJECTSAfter approval from the Ethics Committee of Vienna University School of Medicine and written informed consent were obtained, 40 subjects with AMD were studied. The diagnosis and staging of AMD were based on the results of indirect funduscopy, fundus photography, and scanning laser ophthalmoscopic videoangiography. Visual acuity was determined with Snellen tables. Inclusion criteria were soft or hard drusen of more than 63 µm, hyperpigmentation and/or hypopigmentation of the retinal pigment epithelium, geographic areolar atrophy of the retinal pigment epithelium, or (peri)retinal fibrous scarring. Patients with the exudative form of the disease (retinal pigment epithelial detachments or choroidal neovascular membranes, disciform scarring, or subretinal blood or lipid) were not included in the study. Exclusion criteria were evidence of any other retinal, choroidal, or optic nerve vascular disease; the regular use of pentoxifylline in the past month before the trial period; a limited view of the fundus because of cataract or vitreous hemorrhage; active ocular inflammatory disease; and diabetes mellitus. Only 1 eye of each patient was included according to these criteria. Age-related macular degeneration was classified according to the grading system of Bressler et al.The main subject characteristics are summarized in Table 1.Table 1. Characteristics of Subjects With Age-related Macular Degeneration (AMD)See table graphicSome of the patients took concomitant vasoactive medication because of other diseases. In the pentoxifylline-treated group, 1 patient received a β-adrenoceptor antagonist; 3 patients, calcium channel blockers; 3 patients, angiotensin-converting enzyme inhibitors; and 2 patients, norfenefrine. In the placebo-treated group, 2 patients received β-adrenoceptor antagonists; 4 patients, calcium channel blockers; 5 patients, angiotensin-converting enzyme inhibitors; 1 patient, a β2-adrenoceptor agonist; and 1 patient, theophylline monohydrate. None of these regular medications was discontinued throughout the study.STUDY DESIGNThe study was performed in a double-blind, placebo-controlled, randomized, parallel group design. Subjects were randomly assigned (1:1) to pentoxifylline or placebo treatment. Pentoxifylline (Trental, Albert Roussel Pharma, Vienna, Austria) was administered as an oral dose of 400 mg 3 times a day. Placebo tablets were identical in appearance and taste to maintain the double-blind conditions. Subjects were instructed to take the medication 1 hour after breakfast, lunch, and dinner.STUDY PROTOCOLBaseline measurements of fundus pulsation, laser Doppler flowmetry, and systemic hemodynamics were performed on the first study day. In the morning of the next day, subjects started oral pentoxifylline or placebo treatment. Subjects were readmitted for measurements 1 week, 1 month, 2 months, and 3 months after the start of therapy. A difference of ±2 days was allowed for these follow-up investigations. The measurements were done in the morning before the drugs were taken. Patients' compliance was assessed by tablet count.STUDY METHODSSystolic, diastolic, and mean blood pressures were measured on the upper arm by an automated oscillometric device. Pulse rate was automatically recorded from a finger-pulse oxymetric device (HP-CMS Patient Monitor, Hewlett Packard, Palo Alto, Calif).Pulse synchronous pulsations of the ocular fundus were assessed by laser interferometry. The method is described in detail by Schmetterer et al.Briefly, the eye is illuminated by the beam of a single-mode laser diode with a wavelength (λ) of 783 nm. The light is reflected at both the front side of the cornea and the retina. The reflection from the retina most likely occurs from the Bruch membrane.The 2 reemitted waves produce interference fringes from which the distance changes between the cornea and the retina during a cardiac cycle can be calculated. Distance changes between the cornea and the retina lead to a corresponding variation of the interference order (ΔN[t]). This change in interference order can be evaluated by counting the fringes moving inward and outward during the cardiac cycle. Changes in optical distance (ΔL[t]), corresponding to the distance changes between the cornea and the retina, can then be calculated by ΔL(t) = [ΔN(t) ×λ]/2. The maximum distance change, called fundus pulsation amplitude, estimates the local pulsatile blood flow.The short-term and day-to-day variability of the method is small,which allows even small drug-induced changes in local pulsatile blood flows to be detected.In contrast to systems that record the ocular pressure pulse,information on the ocular circulation can be obtained with high topographic resolution. To obtain information on the choroidal blood flow, the macula, where the retina lacks vasculature, was chosen for the measurements.Retinal microcirculation was assessed with a commercially available scanning laser Doppler flowmeter (Heidelberg Retina Flowmeter, Heidelberg Engineering, Heidelberg, Germany).This system combines laser Doppler flowmetry with laser scanning tomography. Briefly, the vascularized tissue is illuminated by coherent laser light. Scattering on moving red blood cells leads to a frequency shift in the scattered light. In contrast, static scatterers in tissue do not change light frequency, but lead to a randomization of light directions impinging on red blood cells. This light diffusing in vascularized tissue leads to a broadening of the spectrum of scattered light, from which mean red blood cell velocity, the blood volume, and the blood flow can be calculated. These variables are calculated from the backscattered light for each point during the scanning process, and a 2-dimensional map of retinal perfusion is created. These variables can thus be quantified in relative units for any image point. In this study, a 20×20 pixel area was chosen for calculating retinal hemodynamics (200×200 µm). The area was located about 5° nasal to the center of the macula.STATISTICAL ANALYSISThe absolute values were chosen for data analysis. The effect of pentoxifylline on hemodynamic variables was assessed with repeated-measure analysis of variance vs baseline and vs placebo use. Data are presented as mean±SD. A Pvalue of less than .05 was considered the level of significance.RESULTSOf the 40 patients, 33 finished the clinical trial. Three subjects stopped during the first week, 1 subject between the first week and the first month, and 3 subjects between the first and the second months. All 7 patients who discontinued treatment reported drug-related nausea. Of these patients, 4 were in the pentoxifylline group; 3 were in the placebo group.The other 33 patients were included for statistical analysis. In 28 of these patients, the compliance was high, the tablet count being within 5% of the expected value. In 4 other patients, the deviation was 5% to 10%, and in 1 patient it was 12%.During the study, a choroidal neovascular membrane developed in 2 patients, 1 of whom was in the pentoxifylline-treated group, the other in the placebo-treated group. In the other patients, no change was observed in the severity of the disease. Visual acuity was not altered during pentoxifylline or placebo treatment.Baseline ocular hemodynamic values were comparable between the 2 study groups (Figure 1). In the pentoxifylline-treated group, a significant increase in the fundus pulsation amplitude was observed (P<.001 vs baseline and placebo). An increase in the fundus pulsation amplitude was already observed 1 week after the start of pentoxifylline treatment, although the maximum effect occurred at the end of the study (+28%). In contrast, the use of pentoxifylline did not change the retinal blood flow, as evidenced from scanning laser Doppler flowmetry.The effect of 3 months of pentoxifylline treatment (n=16, solid triangles) or placebo (n=17, open triangles) on retinal blood flow (top) and fundus pulsation amplitude (FPA; bottom). Data are presented as mean±SD.Baseline systemic hemodynamic values were comparable between the 2 study groups (Table 2). Neither pentoxifylline nor placebo use had any effect on the mean arterial pressure or the pulse rate.Table 2. Effect of 3 Months of Pentoxifylline or Placebo Use on Mean Arterial Pressure (MAP) and Pulse Rate (Pulse)*See table graphicCOMMENTThe results of this study show that a 3-month therapy with pentoxifylline increases the pulsatile choroidal blood flow in patients with AMD: the fundus pulsation amplitude in the macula increased by more than 25% following the administration of regular oral pentoxifylline, 400 mg 3 times a day. In contrast, we did not observe significant changes in retinal blood flow as evidenced from scanning laser Doppler flowmetry. This is in accordance with previous data in healthy subjects and suggests that pentoxifylline increases choroidal blood flow more than retinal blood flow.Despite this increase in pulsatile choroidal blood flow, we observed no notable increase in visual acuity and no change in the severity of AMD. Given the small number of patients and the short follow-up, this is not surprising. Moreover, the exact role of choroidal perfusion abnormalities in the pathogenesis of AMD has not yet been established. A causal relationship may exist, however, between alterations in choroidal capillary blood flow and diffuse thickening of the Bruch membrane.Whether these changes are initiated by choroidal abnormalities or by the deposition of lipid in the sclera and the Bruch membrane is still a matter of controversy.The use of pentoxifylline increased ocular fundus pulsations, although several of the patients received concomitant vasoactive drugs. The observed increase in pulsatile choroidal blood flow may, therefore, be partly caused by the effects of pentoxifylline on whole blood viscosity, which would be compatible with previous results in healthy subjects.Patients with AMD have been shown not to have altered rheological flow properties.Although we have not assessed variables of blood viscosity in our study, these effects of pentoxifylline may have substantially contributed to our results.Ocular fundus pulsation measurements obviously estimate only the pulsatile component of choroidal blood flow. This does not limit our findings, however, because in this study measurements of the fundus pulsation amplitude rather underestimate the effect of pentoxifylline on total choroidal blood flow. Friedman et alobserved an increased flow pulsatility, as shown by an increased pulsatility index, in the posterior ciliary arteries of patients with AMD. An increase of choroidal blood flow, caused by a reduction in peripheral vascular resistance or a reduction in whole blood viscosity, should lead to a reduction in flow pulsatility. Therefore, the increase in a nonpulsatile choroidal blood flow component might be even larger than that of a pulsatile flow component.The reproducibility of scanning laser Doppler flowmetry is not yet satisfactory. This limits the power of the method to detect drug-induced blood flow changes.Moreover, it has been shown that the relationship between "flow" as obtained with this method and retinal blood flow may not be linear.Nevertheless, results of our previous study indicate that changes of at least 15% over baseline should have been detectable in the present study.Of the 40 subjects, 4 receiving pentoxifylline and 3 receiving placebo discontinued prematurely. This dropout rate is acceptable considering the age of the participants. All 7 subjects who did not finish the trial described nausea after drug administration. This is in agreement with previous reports that gastrointestinal symptoms are the most common adverse effects of the use of pentoxifylline (about 3%), although these and other adverse effects have not occurred to a significantly greater extent than with placebo.A limitation of our results is that we did not measure pentoxifylline plasma levels in the patients with AMD. Subjects' compliance was tested by drug counting, and in all subjects completing the 3-month trial, the tablet count was within 12% of the expected value. This does not ensure that all patients took the medications at regular intervals and at the scheduled times. All measurements, however, were performed in the morning before administering the drug. According to the pharmacokinetics of pentoxifylline,steady-state conditions should have been present at these times. Moreover, a low drug compliance would have resulted in false-negative findings of pentoxifylline's effects.In conclusion, our results indicate that a 3-month treatment with oral pentoxifylline, 400 mg 3 times a day, increases choroidal but not retinal blood flow in patients with AMD. Considering the evidence that choroidal blood flow is impaired in patients with AMD, this result strongly supports the concept that pentoxifylline might be useful in the treatment of this disease. A long-term clinical outcome trial is now warranted to test this hypothesis.NMBresslerSBBresslerSLFineAge-related macular degeneration.Surv Ophthalmol.1988;32:375-413.DPauleikhoffJCChenIHChisholmACBirdChoroidal perfusion abnormality with age-related Bruch's membrane change.Am J Ophthalmol.1990;109:211-217.BPiguetIBPalmvangIHChisholmDMinassianACBirdEvolution of age-related macular degeneration with choroidal perfusion abnormality.Am J Ophthalmol.1992;113:657-663.EFriedmanSKrupskyAMLaneOcular blood flow velocity in age-related macular degeneration.Ophthalmology.1995;102:640-646.PLSonkinSHSinclairDLHatchellThe effect of pentoxifylline on retinal capillary blood flow velocity and whole blood viscosity.Am J Ophthalmol.1993;115:775-780.LSchmettererDKemmlerHBreitenederA randomized, placebo-controlled, double-blind cross-over study of the effect of pentoxifylline on ocular fundus pulsations.Am J Ophthalmol.1996;121:169-176.PLSonkinLWKellySHSinclairDLHatchellPentoxifylline increases retinal capillary blood flow velocity in patients with diabetes.Arch Ophthalmol.1993;111:1647-1652.JSebagMTangSBrownAASadunMACharlesEffects of pentoxifylline on choroidal blood flow in nonproliferative diabetic retinopathy.Angiology.1994;45:429-433.SWolfOArendBBertramHemodilution therapy in central retinal vein occlusion.Graefes Arch Clin Exp Ophthalmol.1994;232:33-39.JKamphuisPSmitsTThienVascular effects of pentoxifylline in humans.J Cardiovasc Pharmacol.1994;24:648-654.JLAmbrusJMAnainSMAnainDose response effects of pentoxifylline on erythrocyte filterability: clinical and animal studies.Clin Pharmacol Ther.1990;48:50-56.PLSonkinSFFreedmanDNeedhamKMKRaoDLHatchellPentoxifylline modulates deformability, F-actin content, and superoxide anion production of polymorphonuclear leukocytes from diabetic cats.Exp Eye Res.1992;55:831-838.LSchmettererFLexerCUnfriedHSattmannAFercherTopical measurement of fundus pulsations.Opt Eng.1995;34:711-716.LSchmettererMWolztASalomonThe effect of isoproterenol, phenylephrine and sodium nitroprusside on fundus pulsations in healthy volunteers.Br J Ophthalmol.1996;80:217-223.CERivaSHarinoBLPetrigRDShonatLaser Doppler flowmetry in the optic nerve.Exp Eye Res.1992;55:499-506.GMichelsonMJLanghansMJMGrohClinical investigation of the combination of a scanning laser ophthalmoscope and laser Doppler flowmeter.Ger J Ophthalmol.1995;4:342-349.GMichelsonBSchmaussMJLanghansJHaraznyMJMGrohPrinciple, validity, and reliability of scanning laser Doppler flowmetry.J Glaucoma.1996;5:99-105.NMBresslerSBBresslerSKWestSLFineHRTaylorThe grading and prevalence of macular degeneration in a Chesapeake Bay waterman.Arch Ophthalmol.1989;107:847-852.WDrexlerCKHitzenbergerHSatmannAFFercherMeasurement of the thickness of fundus layers by partial coherence tomography.Opt Eng.1995;34:701-710.LSchmettererKStrennOFindlEffects of antiglaucoma drugs on ocular hemodynamics in healthy volunteers.Clin Pharmacol Ther.1997;61:583-595.MELanghamKTomeyA clinical procedure for the measurement of the ocular pulse-pressure relationship and the ophthalmic arterial pressure.Exp Eye Res.1978;27:17-25.MELanghamRAFarrellVO'BrienDMSilverPSchilderBlood flow in the human eye.Acta Ophthalmol.1989;67(suppl 191):9-13.LSchmettererMWolztFLexerThe effect of hyperoxia and hypercapnia on fundus pulsations in the macular and the optic disc region.Exp Eye Res.1995;61:685-690.LSchmettererFLexerUGraselliOFindlHGEichlerMWolztThe effect of different mixtures of O2and CO2on ocular fundus pulsations.Exp Eye Res.1996;63:351-355.WInhoffenZNüssgensRheological studies on patients with posterior subretinal neovascularization and exudative age-related macular degeneration.Graefes Arch Clin Exp Ophthalmol.1990;228:316-320.KStrennRMenapaceGRainerOFindlMWolztLSchmettererReproducibility and sensitivity of scanning laser Doppler flowmetry during graded changes in PO2.Br J Ophthalmol.1997;81:360-364.AWardSPClissoldPentoxifylline, a review of its pharmacodynamic and pharmacokinetic properties, and its therapeutic efficacy.Drugs.1987;34:50-97.BBeermanRIngsJMansbyJChamberlainAMc DonaldKinetics of intravenous and oral pentoxifylline in healthy subjects.Clin Pharmacol Ther.1985;37:25-28.ARamesJMPoirierFLeCozPharmacokinetics of intravenous and oral pentoxifylline in healthy volunteers and in cirrhotic patients.Clin Pharmacol Ther.1990;47:354-359.Accepted for publication August 29, 1997.This work was supported in part by grant 6347 from the Oesterreichische Nationalbank, Vienna, Austria.Corresponding author: Leopold Schmetterer, PhD, Department of Clinical Pharmacology, University of Vienna School of Medicine, Währinger Gürtel 18-20, A-1090 Vienna, Austria.

Mueller, Arthur J.; Bartsch, Dirk-Uwe; Folberg, Robert; Mehaffey, Mary G.; Boldt, H. Culver; Meyer, Margaret; Gardner, Lynn M.; Goldbaum, Michael H.; Pe'er, Jacob; Freeman, William R.

1998 JAMA Ophthalmology

doi: 10.1001/archopht.116.1.31pmid: 9445206

ObjectiveTo image the microvasculature of choroidal melanoma with a new confocal scanning laser ophthalmoscope.MethodsEighteen consecutive patients, each with a unilateral choroidal melanoma, were examined prospectively. Indocyanine green angiography was performed with a new confocal scanning laser ophthalmoscope that enabled serial optical sectioning through the tumor. Two additional patients were studied with indocyanine green angiography and confocal scanning laser ophthalmoscopy just before enucleation for posterior choroidal melanomas. The histologic identification of microvasculature patterns was compared with the angiograms for these patients.ResultsIn the series of 18 patients, 16 (89%) indocyanine green angiograms with optical sectioning revealed tubular structures within the melanoma that were identified as tumor vessels based on their angiographic appearance. The microvasculature patterns identified by indocyanine green angiography correlated well with the histologic appearance of these microvasculature patterns in both patients for whom histologic verification was available.ConclusionsThis preliminary study suggests that indocyanine green angiography with confocal scanning laser ophthalmoscopy images the microvasculature of choroidal melanomas and may be capable of detecting microvasculature patterns that have been shown to be prognostically significant from histopathological studies.CHOROIDAL and ciliary body melanomas are among the few forms of cancer that are treated before a pathologist can examine tissue and assign a histologic grade to indicate the likelihood of metastasis. In designing new treatments for these patients, it would be helpful to separate those patients at high risk for metastasis from those at lower risk clinically.Nine microvasculature patterns have been described histologically in choroidal and ciliary body melanomas: normal vessels, avascular zones, straight vessels, parallel straight vessels, parallel vessels with cross-linking, arcs, arcs with branching (incomplete loops), microvascular loops that encircle small microdomains of tumor, and microvascular networks, composed of at least 3 back-to-back closed microvascular loops.In univariate analysis, one pattern, the presence of an avascular (silent) zone, was associated with a favorable prognosiswhereas several other patterns, including arcs, arcs with branching, loops, networks, parallel vessels, and parallel vessels with cross-linking were all associated with metastasis.Melanomas frequently contain combinations of patterns. In multivariate Cox proportional hazards models, 2 histologic microvasculature patterns were strongly associated with death from metastatic melanoma: networks and parallel vessels with cross-linking.Ophthalmic pathologists are now being encouraged to report the presence or absence of microvascular networks and parallel vessels with cross-linking as prognostic factors in diagnostic reports describing eyes removed for malignant melanoma.If it were possible to detect prognostically significant vascular patterns clinically, ophthalmologists might eventually be able to separate patients into histologic risk groups based on these patterns without removing tissue.It is reasonable to suspect that angiography would be capable of detecting microvasculature patterns in vivo. Unfortunately, fluorescein angiography does not show a pathognomonic fluorescence pattern in choroidal melanomas nor are tumor vasculature patterns visualized consistently.Size and pigmentation of the tumor have considerable influence on the appearance of the fluorescein angiogram. The effect of the tumor on adjacent ocular structures, particularly the retinal pigment epithelium, also contributes significantly to its fluorescein angiographic appearance. If the overlying retinal pigment epithelium is completely intact, a fairly normal fluorescein angiogram may result. Overlying retinal vessels are usually visible easily during all phases. However, the so-called double (simultaneous visualization of retinal and choroidal circulation) or tumor circulation is often difficult to recognize and the absence of this finding does not indicate lack of extensive tumor vasculature.Indocyanine green angiography is a relatively new method for imaging choroidal vessels in vivo.Because the near-infrared light used for indocyanine green angiography penetrates the pigmented layers of the fundus more easily than the short wavelength light used in fluorescein angiography, indocyanine green angiography has been primarily used to study leakage of dye in choroidal neovascularization during the late phase and thereby to detect changes in the vascular permeability of these vessels.Some of these early studies also have indicated a possible role for indocyanine green angiography in investigating choroidal masses, but this was not further investigated.Recently, we adapted the confocal scanning laser technology to perform indocyanine green angiography.This technique uses sensitive digital image acquisition and processing. The horizontal image resolution has considerably improved to below 20 µm.The reported histologic microvasculature patterns of prognostic significance fall in this range and should therefore be detectable.This study was designed to evaluate the ability of indocyanine green angiography performed with a new confocal scanning laser ophthalmoscope to image microcirculatory patterns in choroidal melanomas.PATIENTS AND METHODSThis study was divided into 2 phases: a clinical study of the angiographic appearance of the microcirculation with confocal scanning laser ophthalmoscopy and indocyanine green angiography, and an angiographic-histologic correlation in 2 patients.ANGIOGRAPHYIn the first phase, all patients with clear media and with prominent choroidal masses of the posterior pole suspected to be choroidal melanomas who were seen at the University of California, San Diego, Shiley Eye Center between January 1994 and April 1996 were examined prospectively. Diagnosis of choroidal melanoma was established by indirect ophthalmoscopy based on the characteristic appearance of this tumor and confirmed using standardized A- and B-scan ultrasound. Only tumors with a maximum apical height of at least 1.5 mm and low or medium internal reflectivity according to standardized A-scan ultrasound were enrolled. Thereafter, indocyanine green angiography was performed using a confocal scanning laser ophthalmoscope (Heidelberg Retina Angiograph [HRA], Heidelberg Engineering, Heidelberg, Germany). The instrument has been described in detail previously.The optics of this instrument allowed for spherical aberration compensation between −12 and +12 diopters (D). By automatically adjusting the focal plane in steps of 1 D, confocal serial optical sectioning could be obtained. This facilitates visualization of deep tumor vessels. In addition, the tumor height could be measured with this technique by calculating the difference between the confocal plane, in which the apex of the tumor is in focus and the confocal plane, in which the adjacent attached retina is in focus (Figure 1). This method is only valid when performed in areas where the adjacent retina is attached, and, in patients, where the tumor is situated at the posterior pole. Using axial refractive error eye model assumptions for the HRA, the measurement can be converted easily from diopters to millimeters (3 D = 1 mm for emmetropic eyes with an axial correction factor of 2.25% per diopter spherical equivalent for nonemmetropic eyes).For example, if a tumor vessel appears most clearly in a "+6-D confocal plane" in an emmetropic eye, the tumor vessel is located 2 mm anterior to the "0-D starting point" of this series. In a patient with a refraction of +10-D spherical equivalent, the same tumor vessel would be calculated to be 2.45 mm anterior to the "0-D starting point" of this series.Figure 1.Illustration of an optical sectioning series in a tumor of 3 mm height in an emmetropic eye. In this series, the scanning laser beam would be rapidly and consecutively focused in 1-diopter (D) steps from 0 D (tumor basis) to 9 D (tumor apex). Representative steps in this drawing include focusing on the plane of adjacent retina (top, 0 D), on a tumor vessel (middle, 4 D), and on the tumor apex (bottom, 9 D).The confocal indocyanine green angiography sequences were reviewed for the presence of complete serial optical sectioning. Complete serial optical sectioning was defined as including both an optical sectioning at the level of adjacent attached retina and at the level of the tumor apex; such sectioning was performed in 11 patients. Subsequently, maximum tumor height according to serial optical sectioning was corrected for the refraction of the patient, assuming that the refractive error is entirely caused by axial length differences of the eye. The resulting corrected tumor height was compared with ultrasound measurements obtained at corresponding dates.One pixel is equivalent to approximately 33 µm in a 30° angiogram in emmetropic eyes and the lateral correction factor for nonemmetropic eyes is 1.5% per diopter of spherical equivalent.We also validated the pixel counting method in each series by measuring a peripapillary vein, the diameter of which is usually taken to be 125 µm.ANGIOGRAPHIC-HISTOLOGIC CORRELATIONSTwo patients with posterior choroidal melanomas seen at the University of Iowa Hospitals and Clinics, Iowa City, who did not qualify for the Collaborative Ocular Melanoma Study were studied by indocyanine green angiography 2 weeks prior to a scheduled enucleation. Each enucleated eye was fixed in 10% neutral-buffered formalin for at least 48 hours and opened using the alternative gross pathology protocol in which the anterior segment was separated from the posterior pole by a coronal section through the pars plana.This technique permits the pathologist to visualize the surface of the tumor in the same plane as fundus photographer or angiographer and permits precise clinicopathologic correlations and tumor measurements.Each tumor was bisected along the axis of maximum scleral contact and the plane of sectioning was noted on gross photographs. The gross photographs were compared with the indocyanine green angiograms to permit a precise clinicopathologic correlation of angiographic-histologic findings. One half of the tumor block was processed for routine light microscopy in the usual section plane (perpendicular to the surface of the tumor) for purposes of confirming the diagnosis. The other half of the tumor section was embedded and sectioned parallel to the tumor apex (parallel to the sclera) to generate a histologic section plane oriented in the same fashion as the optical cuts through the tumor acquired with HRA. Tumor sections were stained with the modified periodic acid–Schiff stain without hematoxylin counterstaining.This stain correlates well with stains more specific for the endothelium and microcirculation.Histologic sections were digitized and converted to gray-scale images after selecting the green channel to highlight the magenta-stained microcirculation. The resulting image was converted digitally into a "negative," a procedure that makes the microcirculation appear white against a black tumor background for easy histologic-angiographic correlations.RESULTSThe clinical features of the 18 patients examined prospectively by HRA are summarized in Table 1. One patient (patient 7, Table 1) had an amelanotic melanoma. The other tumors were pigmented. In 9 patients, the mass was located partially or completely within the major temporal vascular arcades. In the other 9 patients, the tumor mass was localized entirely outside the arcades. Maximum apical height ranged from 1.5 to 12 mm with a mean (±SD) height of 4.2 (±2.7) mm according to standardized A-scan ultrasound. All patients were untreated at the time the indocyanine green angiography was obtained.Clinical Features of Patients*See table graphicIn 16 (89%) of these 18 patients, visualization of deep tumor vessels was possible by using confocal indocyanine green angiography. The tumor vessels could be identified clearly within the first 30 seconds after injection of dye and fluorescence lasted at least 5 minutes. Thereafter, slow decrease of fluorescence was noted until 10 minutes after injection of dye. Fifteen minutes after injection of dye, no fluorescence within the tumor vessels could be detected and we did not observe late staining in the tumor region in any of our cases. In 2 patients, no tumor vessels could be detected in any confocal plane.In 11 patients, complete serial optical sections during confocal indocyanine green angiography could be used for tumor height measurements. Maximum tumor heights according to these series were calculated between 5 D and 18 D (Table 1). In 7 patients, no serial optical sections could be performed due to subretinal fluid of the adjacent retina. In the 11 tumors with complete serial optical sectioning, the end points of the series included the focal plane at the apex of the tumor as well as the focal plane at the adjacent attached retina. In these patients, the tumor height according to the optical sectioning was corrected according to the spherical equivalent of the patient's refraction (Table 1). All measurements were within 0.25 mm compared with the measurements obtained by standardized ultrasound. For these 11 complete measurements, the correlation coefficient is 0.99 (P<.001) and the mean square root error is 0.11 (Figure 2).Figure 2.Correlation of tumor height measurements according to standardized ultrasound (US) and serial optical sectioning using indocyanine green angiography (ICG).Form, diameter, and localization of the tumor vessels were highly characteristic. In 7 patients, we detected large (132 µm in diameter) elongated vessels with collaterals to other vessels within the tumor (Figure 3). These vessels tended to be present in the center of the tumor and about half the tumor height. In 6 patients, we detected dense convolutions of smaller vessels varying between 66 and 99 µm in diameter (Figure 4) that were located near the tumor surface and at the tumor edges. In 3 patients, we detected tumor vessels measuring 33 µm in diameter or smaller that were elongated and oriented parallel to each other (Figure 5); these vessels could be imaged throughout the whole tumor mass. The tumors of 2 patients contained no vessels (Figure 6): they were angiographically silent. The 2 patients who had angiographic studies before enucleation are described in the following case reports.Figure 3.Patient 4. Choroidal melanoma located in the upper temporal quadrant of the left eye. Maximum height is measured with 4.9 mm according to standardized ultrasound. A, Fundus photograph focused at apex of tumor. Note the localization of retinal vessel bifurcation. B, Fluorescein angiography photograph 39 seconds after injection of dye. No details within the tumor can be seen. C, Optical section using confocal indocyanine green angiography 4 minutes, 33 seconds after dye injection. Confocal plane is taken at about half of maximum tumor prominence (+7 diopters [D] from retinal plane and −8 D from tumor apex). Note localization of retinal vessel bifurcation and compare with photographs A and B. Large elongated vessels, clearly filled with indocyanine green dye, are present within the border of the tumor. D, Confocal section using indocyanine green angiography (16 minutes, 54 seconds after injection of dye). Confocal plane is at same height as in photograph C (+7 D from retinal plane and −8 D from tumor apex). Tumor vessels can no longer be seen. No late staining occurs within the tumor.Figure 4.Patient 7. An amelanotic choroidal melanoma located at the inferior temporal vessel arcade of the right eye. Maximum tumor prominence is measured with 3.1 mm according to standardized ultrasound. A, Fundus photograph. Note localization of overlying retinal vessel. B, Fluorescein angiography photograph 49 seconds after dye injection. Note localization of overlying retinal vessel. Although there is an irregular filling pattern at the surface of the tumor, no double circulation is noted and no deeper tumor vessels are imaged. C, Confocal section using indocyanine green angiography 3 minutes, 6 seconds after injection of dye. Confocal plane is nearly at the apex of the tumor. Compare localization of overlying retinal vessel and border of tumor mass with photographs A and B. Small densely convoluted tumor vessels are located within the tumor borders. D, Confocal section using indocyanine green angiography 3 minutes, 7 seconds after injection of dye. Confocal plane is deeper within the tumor structures. While visibility of retinal vessels are vanishing, visibility of choroidal structures improves. Tumor vessels are getting out of focus. E, Confocal section using indocyanine green angiography 3 minutes, 8 seconds after injection of dye. Confocal plane is nearly at choroidal level. Note the different appearance of normal choroidal vessels and tumor vessels shown in photograph C. F, Confocal section using indocyanine green angiography 3 minutes, 8 seconds after injection of dye. Confocal plane is at choroidal level. The tumor vessels are totally out of focus.Figure 5.Patient 16. Choroidal melanoma located temporally to the macula of the left eye. Maximum prominence is measured with 3.7 mm according to standardized ultrasound. A, Fundus photograph of the melanoma. B, Optical section using confocal indocyanine green angiography 31 seconds after dye injection. Confocal plane is taken at half of maximum tumor prominence (+5 diopters from the retinal plane). Small-diameter parallel vessels are seen within the tumor borders.Figure 6.Patient 6. Choroidal melanoma located inferior to the optic nerve head of the right eye. Maximum prominence is measured with 2.0 mm according to standardized ultrasound. A, Fundus photograph of the melanoma. B, Optical section using confocal indocyanine green angiography 1 minute, 34 seconds after dye injection. Confocal plane is taken at half of maximum tumor prominence (+3 diopters from the retinal plane). No vessels are seen within the tumor borders.REPORT OF CASESCASE 1An 85-year-old woman was seen for a flat, posterior choroidal lesion that she had in her left eye for 16 years. The lesion became elevated over a 3-year period and her visual acuity decreased to 20/200. The patient was excluded from the Collaborative Ocular Melanoma Study because of the presence of an optic nerve pit in the right eye that reduced visual acuity to 20/200. The patient elected to have an enucleation. Two weeks before enucleation, the patient was examined by indocyanine green angiography. The angiogram revealed large diameter vessels (132 µm in diameter) within the tumor on multiple section planes (Figure 7, A). Convoluted vessels and parallel vessels were not identified angiographically. The tumor was bisected vertically, with the temporal half processed for routine histopathology (sections taken perpendicular to the tumor apex) and the nasal half sectioned parallel to the apex and sclera for correlation with the angiogram (Figure 7, B). The tumor was composed of spindle B melanoma cells without evidence of extraocular extension. The histopathologic sections confirmed the presence of large, dilated normal vessels without evidence of parallel vessels with cross-linking, loops, or networks, both in the conventional section plane, and in the plane parallel to the tumor apex, equivalent to the plane of the angiogram (Figure 8). We concluded that the large caliber vessels seen angiographically corresponded to the dilated normal vessels detected histologically on a section plane matched with the angiogram.Figure 7.A, Confocal indocyanine green angiogram from case 1. Note the tubular straight vessels within the choroidal lesion (retinal vessels form an inverted Y superiorly). The broken line indicates the gross section plane. B, Gross photograph of the tumor. Inset shows retinal arterioles pseudocolorized for comparison with angiogram. Broken line indicates gross section plane.Figure 8.Histologic section (case 1) taken in the same plane as the confocal angiogram (parallel to the apex of the tumor), corresponding to tissue to the left of the broken line (Figure 7). Note the large tubular vessels, corresponding to the angiogram. There are no vascular loops or networks (negative photomicrograph, periodic acid–Schiff without hematoxylin counterstaining, ×38).CASE 2A 79-year-old woman with an elevated posterior choroidal mass was referred to University of Iowa Hospitals and Clinics after experiencing a superior visual field defect and decreased visual acuity in the right eye. The patient elected to have an enucleation; she was not eligible for the Collaborative Ocular Melanoma Study because of the large tumor size. Three days before enucleation, the patient was examined by indocyanine green angiography. The angiogram revealed numerous angiographic thin convoluted vessels that formed vascular arcs, arcs with branching, and closed vascular loops (Figure 9, A). The tumor was bisected vertically, with the nasal half processed for routine histopathology (sections taken perpendicular to the tumor apex) and the temporal half sectioned parallel to the apex and sclera for correlation with the angiogram (Figure 9, B). The tumor was composed of spindle B melanoma cells with evidence of infiltration into the sclera around a vortex vein but without extraocular extension. The histopathologic sections confirmed the presence of thin vascular arcs, arcs with branching, and loops but without the formation of networks (defined as at least 3 back-to-back vascular loops; Figure 10).Figure 9.A, Confocal indocyanine green angiogram from case 2. Note the thin-walled vascular arcs and closed back-to-back loops forming a network within the choroidal lesion (retinal vessels form an inverted Y inferiorly). The broken line indicates the gross section plane. B, Gross photograph of the tumor. Inset shows retinal arterioles pseudocolorized for comparison with angiogram. Broken line indicates gross section plane.Figure 10.Histologic section (case 2) taken in the same plane as the confocal angiogram (parallel to the apex of the tumor), corresponding to tissue to the left of the broken line (Figure 9). Note the thin-walled vascular arcs and loops, corresponding to the angiogram (negative photomicrograph, periodic acid–Schiff without hematoxylin counterstaining, ×38).COMMENTIndocyanine green angiography permits visualization of choroidal vasculature as a result of good penetration of absorption and emission light in the near-infrared range through the melanin of retinal pigment epithelium.It has been previously suggested that indocyanine green angiography therefore may have a role in the diagnosis of intraocular tumors, but this was not further investigated at that time.Another more recently published study also investigated the value of indocyanine green angiography in diagnosing and differentiating various choroidal tumors including choroidal melanomas.The authors used a conventional nonscanning and nonconfocal technique. They investigated quantitatively fluorescence intensity changes over time but did not study the visibility of tumor vessels.Because the confocal scanning laser ophthalmoscope we have used allows for confocal serial optical sectioning, the tumor height can be measured with this method by calculating the difference between the lens power, with which the highest and the lowest plane of a prominent mass is in focus.In our pilot study, comparison with ultrasonographic measurements in the 11 patients with complete series revealed that this method is accurate. Thus, we assumed that the same accuracy applies for the localization of microvasculature patterns in the tumor mass.We noted that the diameter of some tumor vessels measured from the indocyanine green angiogram is larger than the diameter of vessels in the histologic microvascular networks or parallel with cross-linking vessels.Electron microscopy studies of these vessels showed severe alterations of the basement membrane as well as gaps in the interendothelial junctions.This could facilitate staining of vessel walls, which would give the vessel a wider appearance in the angiogram than is present histologically. However, if staining of the vessel wall occurred, this would most probably happen significantly later than the appearance of fluorescence inside the blood vessel. In addition, one would also expect late fluorescence of the vessel walls, but we did not observe a change in the diameter of the vessels over time, nor did we observe late staining in any of our patients. In contrast, it is conceivable that the histologic measurements do not reflect the diameter of these vessels in vivo. Collapsing of vessels due to lack of perfusion pressure as well as a "shrinkage" due to fixation and/or preparation steps may alter the lumen of the vessel as imaged with indocyanine green angiography.In the present study, the serial confocal optical sectioning permitted us to detect tumor vessels. Various optical sections through each tumor of 16 patients revealed tubular structures filling with indocyanine green during the early and middle phases. These structures were located within the melanoma borders when compared with fluorescein angiograms and fundus photographs, respectively. The circulation was separate from the retinal circulation and in none of the cases could indocyanine green fluorescence be detected during the late phase in any confocal plane. This indicates that these vessels were not leaking moderate size molecules.For all these reasons, these structures were identified as tumor vessels.The results of this study suggest that indocyanine green angiography and confocal scanning laser microscopy may detect at least some of the microcirculatory patterns described in histologic sections of choroidal and ciliary body melanomas. For example, the large vessels (132 µm in diameter) detected in the tumor described in the first of the 2 case reports corresponded histologically to the normal vascular pattern by means of detailed histologic-angiographic correlations (Figure 3, C, and Figure 7). Additionally, vessels that were convoluted angiographically forming arcs, arcs with branching, and closed vascular loops (Figure 4, C and D) were detected in the tumor of the patient described in the second case report: a careful angiographic-histologic correlation confirmed the identity of the angiographic patterns in tissue sections from corresponding planes (Figure 9and Figure 10). Moreover, the convoluted vessels detected angiographically tended to appear at the periphery and beneath the surface of the tumors, a finding that is significant because microvascular networks in histologic sections of choroidal and ciliary body melanomas tend to form in the same locations.Finally, some tumors did not contain vessels angiographically. Although none of these tumors was available for histologic study, it is possible that these angiographic silent tumors correspond to histologic avascular zones.Our results therefore suggest that further angiographic-histologic correlations are warranted to determine if indocyanine green angiography with confocal scanning laser ophthalmoscopy can be used to detect clinically those histologic tumor vascular profiles that have been associated with more or less favorable prognosis from histologic tissue sections. Indocyanine green angiography with confocal scanning laser ophthalmoscopy may eventually provide a technique by which ophthalmologists can extract information from the noninvasive study of a patient's tumor. 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melanoma.Ophthalmology.1988;95:1576-1582.VRummeltLGardnerRFolbergThree-dimensional relationships between tumor cells and microcirculation using double cyanine-immunolabeling, laser scanning confocal microscopy and computer-assisted reconstruction: an alternative to cast corrosion preparations.J Histochem Cytochem.1994;42:681-686.RFolbergMMehaffeyLGardnerThe microcirculation of choroidal and ciliary body melanomas.Eye.In press.PMontagueMMeyerRFolbergTechnique for the digital imaging of histopathologic preparations of eyes for research and publication.Ophthalmology.1995;102:1248-1251.CShieldsJShieldsPDe PotterPatterns of indocyanine green videoangiography of choroidal tumors.Br J Ophthalmol.1995;79:237-245.DUBartschMIntagliettaJFBilleConfocal laser tomographic analysis of the retina in eyes with macular hole formation and other focal macular diseases.Am J Ophthalmol.1989;108:277-287.HDvorakJNagyJDvorakADvorakIdentification and characterization of the blood vessels of solid tumors that 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Fekrat, Sharon; Goldberg, Morton F.; Finkelstein, Daniel

1998 JAMA Ophthalmology

doi: 10.1001/archopht.116.1.43pmid: 9445207

ObjectiveTo establish a communication between an obstructed retinal vein and the choroid by means of laser in eyes with nonischemic central or branch vein occlusion.MethodsRetrospective review identified eyes with nonischemic central or branch vein occlusion, and with decreasing or persistently decreased visual acuity of 20/100 or worse for 4 months or more before treatment, that received 1 or more sessions of laser photocoagulation to create a chorioretinal anastomosis.ResultsOf 24 eyes with central vein occlusion, an anastomosis formed in 9 (38%) within 2 months after treatment, with visual improvement of 6 or more lines in 2 (8%) of 24 eyes, 1 to 3 lines in 5 (21%), and no improvement in 2 (8%). Of 6 eyes with branch vein occlusion, an anastomosis formed in 3 (50%) within 2 months after treatment, with visual improvement of 1 to 3 lines in 2 (33%) of 6 and no improvement in 1 (16%). No permanent, vision-limiting complications occurred during a mean follow-up of 13 months after the first treatment session or 8 months after the last session.ConclusionsLaser photocoagulation of a retinal vein and Bruch's membrane may create a chorioretinal anastomosis in some eyes with a nonischemic vein occlusion. Progression to an ischemic status may possibly be prevented with successful anastomosis formation. Marked visual improvement may occur. Treatment techniques to create reliably an anastomosis with subsequent visual improvement, while minimizing potential complications, continue to evolve.RETINAL VEIN occlusion is a common cause of visual loss. Until recently, treatment options for affected eyes have been directed at management of sequelae of the venous occlusion,including macular edema and neovascularization, by grid or scatter-type photocoagulation, and have not been aimed at reestablishing the venous outflow of the retina. A chorioretinal anastomosis between a retinal vein and the choroid may bypass the occluded vein and relieve the venous obstruction. This may decrease the conversion rate of a nonischemic vein occlusion to an ischemic status and lessen macular edema with concurrent improvement in visual acuity.Attempts to create a chorioretinal venous anastomosis by means of laser photocoagulation have been successful in some eyes.Of 24 eyes with a nonischemic central retinal vein occlusion (CVO) treated in this fashion by McAllister and Constable,8 (33%) developed an anastomosis within 7 weeks after treatment coincident with some visual improvement; 3 (12%) of 24 had marked visual improvement of 6 lines or more. None of these 8 eyes progressed to an ischemic status. In the Central Vein Occlusion Study, one third of eyes with perfused CVO progressed to a nonperfused status during follow-up, occurring most frequently in the first 4 months.Creating an anastomosis in eyes with branch retinal vein occlusion (BVO) has not been reported.We describe 30 eyes of 30 patients with a nonischemic vein occlusion (24 with CVO and 6 with BVO) who underwent laser photocoagulation during 1 or more treatment sessions in an attempt to create a chorioretinal anastomosis.PATIENTS AND METHODSPATIENTSThe Wilmer Retinal Vascular Center patient records, Wilmer Ophthalmological Institute, Baltimore, Md, were retrospectively reviewed to identify consecutive patients with ophthalmoscopic and angiographic evidence of a nonischemic CVO or BVO in at least 1 eye of at least 4 months' duration (CVO: mean, 12 months; range, 4-26 months; BVO: mean, 16 months; range, 6-24 months) and who received 1 or more sessions of laser photocoagulation, in an attempt to create a chorioretinal anastomosis from December 1994 to May 1996. Each patient must have demonstrated decreasing or persistently decreased visual acuity of 20/100 or less in the affected eye because of perfused macular edema during at least a 4-month interval before the first treatment session (CVO: mean, 10 months; range, 4-26 months; BVO: mean, 14 months; range, 6-23 months). Twenty-four treated patients with a nonischemic CVO and 6 treated patients with a nonischemic BVO were identified.TREATMENT TECHNIQUEAfter discussing the risks, benefits, and alternatives to the creation of a laser-induced chorioretinal anastomosis, we obtained written, informed consent from each patient. With the use of topical anesthesia, a fundus contact lens, and a slitlamp delivery system, laser photocoagulation was applied to selected locations along a retinal vein, usually about 2 disc diameters nasal to the optic nerve in eyes with CVO and within 1 disc diameter peripheral to the occlusion site in eyes with BVO. The laser photocoagulation treatment spot was positioned on the edge of or directly on top of a retinal vein at all treatment sessions, except at 1 session, in which 1 spot was used to treat the adjacent Bruch's membrane first, followed by the vein itself (see case 2 below). Signs of presumed rupture of Bruch's membrane, such as a vaporization bubble, and occasionally a small, self-limited intravitreal stream of hemorrhage, presumably from the adjacent retinal vein, were sought. Various laser parameters and wavelengths, including argon green, argon blue-green, and dye yellow, were used. Powers of 3.0 W were used in some cases. In all cases, the spot size was 50 µm, and the duration was 0.1 second. At some treatment sites, the Nd:YAG system was solely used with an energy setting of about 4 mJ.For 30 eyes (24 with CVO and 6 with BVO), 57 treatment sessions were completed with a mean of 1.9 treatment sessions per patient (range, 1-4 sessions). Laser photocoagulation treatment parameters differed, because the superiority of 1 combination of parameters compared with another had not been previously demonstrated. Specifically, argon blue-green wavelength with 3.0 W of power and a 50-µm spot size and 0.1-second duration was used in 25 (44%) of the 57 treatment sessions; dye yellow wavelength with 700 to 800 mW of power, a 50-µm spot size and 0.1-second duration was used in 13 (23%) of the 57 sessions; argon green or blue-green wavelengths with 0.8 to 1.5 W, 50 µm, and 0.1 second were used in 11 (19%) of the 57 sessions; Nd:YAG laser was used in the remaining 8 sessions (14%). Each laser, including the Nd:YAG laser, was used alone and not as an adjunct to the other lasers.The patient was followed up at monthly intervals after treatment to monitor for the development of an anastomosis. The primary criterion used to determine whether a chorioretinal anastomosis had formed during follow-up was the ophthalmoscopic appearance of the central and/or peripheral segment of the treated retinal vein dipping down into the choroid at a treatment site.If the initial treatment session was unsuccessful in producing a chorioretinal venous anastomosis after at least 3 months of follow-up, a second treatment session was offered to the patient with the use of the same technique at previously untreated sites. Pretreatment and posttreatment fundus photographs and fluorescein angiography were obtained. During follow-up, progression of the nonischemic vein occlusion to an ischemic status was defined by the development of more than 10 disc areas of capillary nonperfusion on fluorescein angiography.RESULTSCENTRAL RETINAL VEIN OCCLUSIONPretreatment characteristics of the 24 eyes of 24 patients with a nonischemic CVO are outlined in Table 1, with subgroup analysis of the eyes that developed an anastomosis and those that did not.Table 1. Pretreatment Characteristics in Eyes With Nonischemic CVO*See table graphicNine (38%) of the 24 treated eyes developed a chorioretinal anastomosis within 8 weeks after treatment. Of these 9 eyes, the mean duration of decreasing or persistently decreased visual acuity to a level of 20/100 or worse because of perfused macular edema before the first treatment was 9 months. One eye (11%) had received grid laser photocoagulation for cystoid macular edema at least 4 months before the first anastomosis attempt, whereas 1 (11%) had the grid treatment at the same time as the first anastomosis attempt. The grid treatment did not result in improved visual acuity in either eye. None of the 9 eyes demonstrated progression to an ischemic status during a mean follow-up of 11 months after the first treatment session. Of the 15 eyes (62%) that did notdevelop an anastomosis, an ischemic status developed in 3 (20%) during a mean follow-up of 14 months after the first treatment session.Eighteen treatment sessions, in an attempt to create a chorioretinal anastomosis, were completed for the 9 patients who developed an anastomosis, with a mean of 2 treatment sessions per patient (range, 1-4 sessions) (Table 2). Of the 18 sessions, only the last treatment session for each of the 9 eyes (9 sessions) resulted in a successful anastomosis. For the 3 eyes that required only 1 treatment session to create an anastomosis, the mean follow-up was 9 months. For the 4 eyes that required a second treatment session to create an anastomosis, the mean follow-up after the second session was 3 months. For the 1 eye that required a third treatment session to create an anastomosis, the follow-up after the third session was 4 months. For the 1 eye that required a fourth treatment session to create an anastomosis, the follow-up after the fourth session was 4 months.Table 2. Treatment and Posttreatment Characteristics in Eyes With Nonischemic CVO*See table graphicOf the 9 sessions that resulted in an anastomosis in these 9 eyes, the laser photocoagulation treatment parameters varied. Of the 9 sessions, 3.0 W of argon blue-green (using a 50-µm spot size and 0.1-second duration) resulted in anastomosis formation in 8 of these sessions (89%). In the remaining 1 session for 1 eye, 700 mW of dye yellow at 50 µm and 0.1-second duration produced a chorioretinal anastomosis.Of the 9 eyes that developed an anastomosis (Figure 1), 2 had marked and prompt visual improvement of 6 or more lines (2 of 24 [8]), 5 had some visual improvement of 1 to 3 lines (5 of 24 [21]), and 2 (2 of 24 [8]) had no visual improvement within 8 weeks after the treatment session that led to anastomosis formation, which may have resulted from the presence of foveal retinal pigment epithelial hyperpigmentation, before treatment, associated with chronic cystoid macular edema.Figure 1.Visual acuity (VA) at first treatment session to create an anastomosis vs final visual acuity in eyes with nonischemic central retinal vein occlusion. Points above the line represent deterioration in visual acuity; points below the line, improvement; and points on the line, unchanged. HM indicates hand motions; CF, counting fingers.For descriptive purposes only, the ophthalmoscopic appearance of the chorioretinal anastomosis was categorized into 1 of 3 groups, based on the diameters of the central and peripheral venous segments emanating from the anastomotic site. One group (Figure 2) consisted of those anastomoses where the central venous segment had a relatively larger diameter than the relatively smaller diameter of the peripheral venous segment. Five of the 9 eyes that developed an anastomosis had this configuration. Another group consisted of those anastomoses where the central venous segment was relatively smaller in caliber than the peripheral venous segment; in fact, the central segment was markedly narrowed and even absent ophthalmoscopically in 1 eye (Figure 3). Four of the 9 eyes that developed an anastomosis had this configuration. In the 1 eye that developed 2 chorioretinal anastomoses (case 1 below), the ophthalmoscopic appearance of one of the anastomoses may be categorized into a third group in which the central segment is of the same caliber as the peripheral segment.Figure 2.Five of 9 eyes with nonischemic central retinal vein occlusion developed an anastomosis where the central venous segment leading to the anastomosis had a relatively larger diameter (arrow) than the smaller diameter of the peripheral venous segment (curved arrow). Both segments dipped down toward the choroid.Figure 3.Four of 9 eyes with a nonischemic central retinal vein occlusion developed an anastomosis where the peripheral venous segment leading from the anastomosis had a relatively larger diameter (arrow) than the markedly narrow and almost involuted central segment (curved arrow). The peripheral segment dipped down toward the choroid. The central segment dipping down into the choroid was difficult to observe when the segment was almost involuted.No permanent, vision-limiting complications were encountered in any of the 24 eyes during a mean follow-up of 13 months after the first treatment session or 7 months after the most recent treatment session (Table 3). Eight eyes (33%) did not develop any complications. Of the 16 eyes (67%) that developed a complication of treatment, the severity varied, and 2 of the 16 eyes developed more than 1 complication. A transient vitreous hemorrhage occurred in 10 eyes (10 of 24 eyes [42]) at the time of treatment, 9 of which had received only Nd:YAG laser photocoagulation at 1 or more treatment sites. The hemorrhage was mild in all 10 eyes immediately after treatment and had cleared in all eyes at the 2-month follow-up visit; no eyes were examined before the 2-month follow-up visit. Localized choroidal neovascularization developed in 5 eyes (5 of 24 eyes [21]) and did not affect visual acuity or require treatment in any instance at the most recent follow-up. Preretinal fibrosis developed in 3 eyes (3 of 24 eyes [12]). A vitrectomy was performed in 1 of these 3 eyes to release a secondary, small, localized traction retinal detachment. No eye developed segmental retinal ischemia or choroidovitreal neovascularization in this series.Table 3. Complications in Eyes With Nonischemic CVO During Follow-up*See table graphicBRANCH RETINAL VEIN OCCLUSIONPretreatment characteristics of the 6 eyes of 6 patients with a nonischemic BVO are outlined in Table 4, with subgroup analysis of the eyes that developed an anastomosis and those that did not.Table 4. Pretreatment Characteristics in Eyes With Nonischemic BVO*See table graphicThree (50%) of the 6 treated eyes developed a chorioretinal anastomosis within 8 weeks after treatment. Of the 3 eyes with an anastomosis, the mean duration of decreasing or persistently decreased visual acuity to a level of 20/100 or worse because of perfused macular edema before the first treatment was 13 months. Two of these eyes (67%) had received grid laser photocoagulation for cystoid macular edema at least 4 months before the first anastomosis attempt. The grid treatment did not lead to improvement in visual acuity in either eye. None of the 6 eyes demonstrated progression to an ischemic status during a mean follow-up of 13 months after the last treatment session.Of the 3 eyes that developed an anastomosis, each had received only 1 treatment session (Table 5). Follow-up for the 3 eyes was 11 months (range, 4-16 months) after the first and only treatment session. Laser photocoagulation treatment parameters varied. At 2 (67%) of the 3 sessions, the argon blue-green wavelength using 3.0 W of power at 50 µm and 0.1-second duration was used. At the remaining 1 session for 1 eye (33%), 1.0 W of argon green at 50 µm and 0.1-second duration was used.Table 5. Treatment and Posttreatment Characteristics in Eyes With Nonischemic BVO*See table graphicOf the 3 eyes (50%) that developed an anastomosis, 2 had some visual improvement of 1 to 3 lines (2 of 6 [33]), and 1 had no visual improvement (Figure 4), which may have been caused, in part, by the presence of foveal pigment associated with chronic cystoid macular edema. In all cases, both the central and peripheral venous segments appeared to dip down into the choroid. The central venous segment was relatively smaller in caliber than the peripheral segment in all 3 cases.Figure 4.Visual acuity (VA) at first treatment session to create an anastomosis vs final visual acuity in eyes with nonischemic branch retinal vein occlusion. Points above the line represent deterioration in visual acuity; points below the line, improvement; and points on the line, unchanged. HM indicates hand motions; CF, counting fingers.No permanent, vision-limiting complications were encountered in the 6 treated eyes during a mean follow-up of 15 months after the first treatment session or 13 months after the most recent treatment session (Table 6). Three eyes (50%) did not develop any complications. In the 3 eyes (50%) that developed a complication of treatment, a transient vitreous hemorrhage was encountered in 1 eye (1 of 6 eyes [17]) that received only Nd:YAG laser photocoagulation at 1 or more treatment sites. The hemorrhage was mild at the time of treatment and had resolved by the 2-month follow-up visit; the patient was not examined before the 2-month follow-up visit. In the other 2 eyes (2 of 6 eyes [33]), mild, preretinal fibrosis developed localized to the treatment site. No treated eye with a BVO developed choroidal neovascularization, segmental retinal ischemia, or choroidovitreal neovascularization.Table 6. Complications in Eyes With Nonischemic BVO During Follow-up*See table graphicREPORT OF CASESCASE 1A 53-year-old healthy white man came to The Wilmer Retinal Vascular Center in March 1995 with a 12-month history of a nonischemic CVO in the right eye. Corrected visual acuity was 20/100 OD and 20/20 OS, and results of pupillary, intraocular pressure, and slitlamp examinations were normal. No neovascularization of the iris or angle was present. Ophthalmoscopy of the right eye disclosed disc edema, cystoid macular edema, mild venous tortuosity, and rare intraretinal hemorrhage (Figure 5, A). Fluorescein angiography confirmed the nonischemic nature of the CVO (Figure 5, B) with cystic parafoveal fluorescein leakage in the late-phase frames (Figure 5, C). Ophthalmoscopy of the left eye was normal.Figure 5.A, On initial examination, ophthalmoscopy confirmed the 12-month history of a nonischemic central retinal vein occlusion with disc edema, cystoid macular edema with some intracystic hemorrhage, mild venous tortuosity, and rare intraretinal hemorrhage. Note the choroidal vessel (arrow) (compare with E). B, Fluorescein angiography confirmed the nonischemic status of the occlusion. Note the patchy choroidal filling inferonasal to the optic nerve head (arrow) (compare with I). C, Cystic parafoveal fluorescein leakage (arrow) was present in the late-phase frames. Fluorescein leakage at the optic disc was also present. D and E, One application of laser photocoagulation was placed overlying the retinal vein in an attempt to puncture Bruch's membrane and the retinal vein with 1 spot at each treatment site. E, Inferonasally, treatment was placed along the retinal vein where an underlying choroidal vessel was noted (compare with A). F, Seven weeks after treatment, the disc edema and macular edema improved (compare with A). In some areas, the venous tortuosity had also improved. A chorioretinal anastomosis was noted ophthalmoscopically at both treatment sites (arrows). Superonasally, the central venous segment had a relatively larger diameter than the peripheral venous segment; both dipped down toward the choroid. Minimal fibrosis was present at the treatment site. Inferonasally, the central venous segment was of the same diameter as the peripheral venous segment; both dipped down toward the choroid. G, Fluorescein angiography disclosed earlier venous filling within the central venous segment (straight arrows) compared with the peripheral venous segment (curved arrows) at both treatment sites. H, Seven months after treatment, further resolution of the venous tortuosity and disc hyperemia had occurred (compare with A). The morphological configuration of the inferonasal anastomosis had changed during follow-up (large arrow) (compare with F). The nearby choroidal vessel, noted before treatment (see A), had an enlarged diameter, suggesting increased flow within it. I, Fluorescein angiography of the inferonasal anastomosis demonstrated both the central and peripheral (straight arrows) venous segments joining into a common venous trunk that dipped down toward the choroid through a venous loop (curved arrow). The more prominent filling within the venous loop, compared with each venous trunk, may be attributed to the larger volume of blood filling the loop, supplied from 2 trunks. Patchy choroidal filling, which was present in this area before treatment (compare with B), was again noted. J, Although parafoveal fluorescein leakage remained in the late-phase frames (arrow), the macula was less cystic and thickened.Laser photocoagulation with the argon blue-green wavelength, 3.0 W, 50-µm spot size, and 0.1-second duration was applied about 2 disc diameters superonasal and inferonasal to the optic nerve. One spot was placed in each location overlying the retinal vein to puncture the retinal vein and, hopefully, subjacent Bruch membrane with the 1 spot (Figure 5, D and E). Inferonasally, treatment was applied to the retinal vein where an underlying choroidal vessel was noted (Figure 5, A and E). No bleeding was encountered.Seven weeks later, visual acuity had improved to 20/30 OD with subjective visual improvement. Ophthalmoscopy disclosed resolution of the disc edema with lessened macular edema and venous tortuosity (Figure 5, F). A chorioretinal anastomosis was noted ophthalmoscopically at both treatment sites. The presence of an anastomosis was suggested at each site by the ophthalmoscopic appearance of the central and peripheral segments of the treated retinal vein dipping down into the choroid. Superonasally, the central venous segment had a relatively wider diameter than the peripheral segment of the same vein (Figure 5, F). Inferonasally, both the central and peripheral segments of the retinal vein were of approximately the same caliber (Figure 5, F). Fluorescein angiography disclosed earlier venous filling in the central venous segment compared with the peripheral venous segment at both treatment sites (Figure 5, G). Trilaminar venous flowwas not observed.Seven months after treatment, visual acuity was 20/25 OD. Further resolution of the cystoid macular edema and venous tortuosity had occurred (Figure 5, H). The morphological configuration of the inferonasal anastomosis had changed (Figure 5, H and I). Fluorescein angiography demonstrated both the central and peripheral venous segments joining into a common venous trunk that dipped down toward the choroid through a venous loop (Figure 5, I). Although parafoveal fluorescein leakage remained in the late-phase frames, the macula was less cystic and thickened (Figure 5, J). No complications occurred.CASE 2A 56-year-old white man with systemic hypertension came to The Wilmer Retinal Vascular Center in November 1994 with an 11-month history of a nonischemic CVO in the right eye. Corrected visual acuity was 20/60 OD and 20/20 OS. Results of pupillary, intraocular pressure, and slitlamp examinations were normal. No neovascularization of the iris or angle was present. Ophthalmoscopy of the right eye disclosed disc congestion, marked macular edema with central cyst formation, mild venous tortuosity and engorgement, and rare intraretinal hemorrhage. Fluorescein angiography confirmed the nonischemic nature of the CVO and demonstrated parafoveal dye leakage in the late-phase frames. Ophthalmoscopy of the left eye was normal. Grid laser photocoagulation was applied to the superior sector of the cystoid macular edema in the right eye, by means of argon green, 50 µm, 0.1-second duration, and 100 mW of power, without complication.Four months later, the visual acuity had decreased to 20/100 OD. Ophthalmoscopy was essentially unchanged (Figure 6, A). Fluorescein angiography disclosed a nonischemic CVO with marked dye leakage accumulating parafoveally in a cystic pattern in the late-phase frames (Figure 6, B). In an attempt to create a chorioretinal anastomosis, 700 mW of dye yellow laser with a 50-µm spot size and 0.1-second duration was used to puncture first Bruch's membrane and then the adjacent retinal vein in 8 locations for a total of 16 bursts, 2 at each treatment site. One of the chosen treatment sites was where the superonasal vein appeared to dip down into the neurosensory retina (Figure 6, A).Figure 6.A, Four months after initial examination, ophthalmoscopy demonstrated no change in the disc congestion and mild venous tortuosity. Despite the superior grid treatment, the cystic macular edema remained with a large, central cyst (large arrow). Note the short segment of the superonasal retinal vein that appears to disappear into the retina and then immediately reappear (small arrow) (compare with C). B, Fluorescein angiography demonstrated cystic parafoveal fluorescein accumulation in the late-phase frames. C, About 2 months after treatment, a chorioretinal anastomosis had formed superonasally (arrow) (compare with A). The disc congestion and cystoid macular edema had improved (compare with A). D, Fluorescein angiography demonstrated laminar venous flow in the larger-diameter central vein (straight arrow) compared with less laminar flow in the narrower peripheral vein (curved arrow) in this angiographic frame. E, Two months after treatment, parafoveal fluorescein dye leakage remained, but it was less cystic and thickened. Although the disc remained hyperfluorescent, the associated dye leakage had lessened. F, Indocyanine green angiography suggested drainage from the anastomosis into a choroidal vessel (arrows), presumably a choroidal vein.Two months after treatment, the visual acuity had improved to 20/50 OD with marked subjective improvement. The disc congestion, venous tortuosity, and cystoid macular edema had improved. A chorioretinal anastomosis was present superonasally (Figure 6, C) and was suggested by the ophthalmoscopic appearance of the central and peripheral segments of the treated retinal vein dipping down into the choroid. The central segment had a relatively wider diameter than the peripheral segment. An anastomosis did not develop at any of the 7 other treatment sites. Fluorescein angiography demonstrated laminar venous flow in the central, wider vein compared with relatively less laminar flow in the peripheral, narrower vein (Figure 6, D). Trilaminar venous flowwas not observed. Although parafoveal fluorescein accumulation was present in the late phase of the angiogram, the macula was less cystic and thickened (Figure 6, E). Indocyanine green angiography (Figure 6, F) suggested drainage from the anastomosis into a choroidal vessel, presumably a choroidal vein; however, given the other prominent choroidal vessels in the vicinity, the finding was only suggestive.Nineteen months after treatment, the visual acuity was 20/25 OD. The ophthalmoscopic and angiographic appearance of the superonasal anastomosis remained unchanged. No complications occurred.COMMENTInstead of managing the consequences of the vein occlusion such as macular edema and neovascularization by grid or scatter-type photocoagulation,treatment modalities have recently been aimed at restoring venous flow by bypassing the occlusion altogether.The concept of creating a chorioretinal anastomosis between a retinal vein and the choroid was initially introduced almost 50 years ago by Verhoeffwhen he described such an anastomosis that developed after diathermy. During the following 40 years, the idea resurfaced within the literature several timesand was most recently popularizedas a potential therapeutic modality for eyes with a nonischemic CVO by bypassing the occluded vein, thereby relieving the obstruction. The rationale for attempting to reestablish venous outflow after creating a chorioretinal anastomosis has been to alter the natural courseby improving visual acuity, as associated macular edema decreases, and by decreasing the conversion rate of the vein occlusion to an ischemic status.Performing a retinal vein bypass in eyes with an ischemicvein occlusion has not been widely attempted, because reestablishing venous outflow in these eyes would not likely lead to either reperfusion of the areas of retinal capillary dropout or improved visual acuity. However, if the parafoveal and perifoveal areas remain nonischemic in an eye with an otherwise largely ischemic CVO, there may be some visual benefit from improved venous outflow and lessened macular edema. Reperfusion of the remaining retinal capillaries may also lessen the risk of developing rubeosis. Since ischemic eyes might be more likely to develop the fibrovascular complications from attempting to create a laser-induced chorioretinal anastomosis (I. L. McAllister, FRACO, oral communication, 1997), such as choroidovitreal neovascularization, further investigation is needed before this treatment approach is recommended for ischemic eyes.Laser parameters and treatment techniques for reproducible anastomosis formation in eyes with nonischemic vein occlusion have not yet been determined. It is not clear from our study why an anastomosis developed in only 9 (38%) of 24 eyes with CVO and only 3 (50%) of 6 eyes with BVO. Ophthalmoscopic determination of whether the retinal vessel or Bruch's membrane was definitely punctured by the laser application may be impossible. Perhaps 1 wavelength, such as argon blue-green, argon green, dye yellow, or Nd:YAG, is superior to the others, or perhaps using 1 in combination with another may prove to be preferable.The necessary power to successfully produce an anastomosis is also unknown. Of the 12 eyes that developed an anastomosis, 10 (83%) had been treated with the relatively high power of 3.0 W with the use of the argon blue-green wavelength at the session that led to anastomosis formation. Of the remaining 2 eyes, 1 was treated with 700 mW of dye yellow and the other with 1.0 W of argon green.Factors other than power and wavelength may possibly determine whether or not an anastomosis will form, such as whether an underlying choroidal vein is near the treated area (Figure 5, A and E), whether a segment of the pretreatment retinal vein dips into the neurosensory retina at the treatment site (Figure 6, A and C), the age of the patient, the presence of collateral vessels on the optic nerve head, media clarity to facilitate treatment, and the degree of intravascular pressure elevation in the obstructed vein, among others. However, if creation of an anastomosis is attempted earlier in the course of the occlusive process, perhaps when the intravascular pressure is most elevated, the outcome may be clouded by the natural course; a control population would be necessary.It may be that some eyes respond to the injury of laser photocoagulation with anastomosis formation, whereas others react with a fibrovascular response involving much smaller vessels.It has also not yet been determined whether puncture of both the retinal vein and Bruch's membrane is necessary for anastomosis formation; it may be that puncture of only 1 of these structures, such as Bruch's membrane, is sufficient. However, puncture of Bruch's membrane first, followed immediately by puncture of the adjacent vein, or puncture of both the retinal vein and subjacent Bruch's membrane with 1 spot, may be preferable. Further investigation is necessary.The ophthalmoscopic and angiographic criteria necessary to determine the presence of a chorioretinal anastomosis have not been well described. Trilaminar venous flowhas been suggestive of anastomosis formation; however, this was not observed in our patients. Unfortunately, analyses of the fluorescein angiograms in our 30 patients did not help to determine whether blood was preferentially flowing into the anastomosis. In our study, the primary criterion used to determine whether a chorioretinal anastomosis had formed during follow-up was the ophthalmoscopic appearance of the central and/or peripheral segment of the treated retinal vein dipping down into the choroid at a treatment site.In our series of 30 eyes, an anastomosis formed in 12 eyes (40%): 9 (38%) of 24 with nonischemic CVO and 3 (50%) of 6 with nonischemic BVO. Even when an anastomosis was produced from the laser application, however, it led to marked visual improvement (≥6 lines) only in 2 eyes (8%) with nonischemic CVO. In these 2 eyes, it is unlikely that the visual improvement reflected the natural course of the CVOsince, in both cases, decreased vision had been present for about 1 year, with prompt (within 8 weeks) and marked visual benefit and obvious decompression of the venous tree after creation of the laser-induced anastomosis. The fact that only 2 eyes with an anastomosis had marked visual improvement, compared with 10 eyes with an anastomosis that did not have marked visual improvement, may result from a variety of factors that may include pretreatment visual acuity, pretreatment presence of foveal pigment or degeneration, the presence or absence of previous extensive grid laser photocoagulation, ophthalmoscopic type of chorioretinal anastomosis that formed (see the "Results" section; Figure 2, Figure 3, and Figure 5, F), and other presently unrecognized factors.Among the 12 eyes that developed an anastomosis, a relative difference in venous caliber between the central and peripheral venous segments at the anastomotic site was observed. Whether this observation is related to the amount of blood flow within the venous segment and, consequently, the effectiveness of the anastomosis is not known. Nevertheless, we categorized the ophthalmoscopic appearance of the chorioretinal anastomosis into 3 groups, based on the diameters of the central and peripheral venous segments emanating from the anastomotic site; there may be no consistent relationship between venous caliber and functional success of an anastomosis. The implications of these observations require further investigation.Although there were no permanent, vision-limiting complications in our series of 30 eyes, we remain concerned about the development of such complications after this procedure, and we are therefore presently willing to treat only those eyes whose visual acuity is 20/100 or worse and who have been followed up for at least 4 months with decreased or decreasing visual acuity. Potential treatment complications include long-standing, vision-limiting hemorrhage,preretinal fibrosis with or without associated traction and/or rhegmatogenous retinal detachment,choroidal neovascularization,segmental retinal ischemia, and choroidovitreal neovascularization.Laser perforation of a retinal vein and underlying or adjacent Bruch membrane may successfully create a chorioretinal anastomosis in some eyes and may offer improved visual status and perhaps a decreased risk of progression to an ischemic status in eyes with a nonischemic CVO or BVO. The precise treatment variables and technique to reliably create an anastomosis, while minimizing potential complications, continue to evolve.Branch Vein Occlusion Study GroupArgon laser photocoagulation for macular edema in branch vein occlusion.Am J Ophthalmol.1984;98:271-282.Branch Vein Occlusion Study GroupArgon laser scatter photocoagulation for prevention of neovascularization and vitreous hemorrhage in branch vein occlusion: a randomized clinical trial.Arch Ophthalmol.1986;104:34-41.Central Vein Occlusion Study GroupEvaluation of grid pattern photocoagulation for macular edema in central vein occlusion: the central vein occlusion study group M report.Ophthalmology.1995;102:1425-1433.Central Vein Occlusion Study GroupA randomized clinical trial of early panretinal photocoagulation for ischemic central vein occlusion: the central vein occlusion study group N report.Ophthalmology.1995;102:1434-1444.ILMcAllisterIJConstableLaser-induced chorioretinal venous anastomosis for treatment of nonischemic central retinal vein occlusion.Arch Ophthalmol.1995;113:456-462.Central Vein Occlusion Study GroupBaseline and early natural history report: the central vein occlusion study.Arch Ophthalmol.1993;111:1087-1095.Central Vein Occlusion Study GroupNatural history and clinical management of central retinal vein occlusion.Arch Ophthalmol.1997;115:486-491.FHVerhoeffSuccessful diathermy treatment in a case of recurring retinal hemorrhages and retinitis proliferans.Arch Ophthalmol.1948;40:239-244.BAKlienSpontaneous vascular repair: arteriolar and venous cilioretinal communications.Am J Ophthalmol.1960;50:691-701.WCKennethAnastomoses between the retinal and ciliary arterial circulations.Br J Ophthalmol.1956;40:65-81.PAmalricPBessouFLescureThe problems of cilioretinal anastomoses in central vein thromboses: therapeutic deduction.Bull Soc Ophthalmol Fr.1961;11:882-888.EDWolfMFGoldbergChorioretinal vascular anastomoses resulting from photocoagulation in cynomolgus monkeys.Ophthalmic Surg.1980;11:30-38.ILMcAllisterD-YYuSVijayasekaranCBarryIConstableInduced chorioretinal venous anastomosis in experimental retinal branch vein occlusion.Br J Ophthalmol.1992;76:615-620.SVijayasekaranD-YYuIMcAllisterCBarryIConstableOptimal conditions required for the creation of an iatrogenic chorioretinal venous anastomosis in the dog using argon green laser photocoagulation.Curr Eye Res.1995;14:63-70.DFinkelsteinJGClarksonRetinal vessel bypass: a promising new clinical investigative procedure.Arch Ophthalmol.1995;113:421-422.SGEccariusMJMoranJGSlingsbyChoroidal neovascular membrane after laser-induced chorioretinal anastomosis.Am J Ophthalmol.1996;122:590-591.S-SYarngC-LHsiehChoriovitreal neovascularization following laser-induced chorioretinal venous anastomosis.Ophthalmology.1996;103(suppl):136. Abstract.DJBrowningMHRotbergVitreous hemorrhage complicating laser-induced chorioretinal anastomosis for central retinal vein occlusion.Am J Ophthalmol.1996;122:588-589.JKLuttrullEpiretinal membrane and traction retinal detachment complicating laser-induced chorioretinal venous anastomosis.Am J Ophthalmol.1997;123:698-699.Accepted for publication July 14, 1997.Supported in part by the Ronald G. Michels Fellowship Foundation, Baltimore, Md; Heed and Heed/Knapp Fellowship Foundation, Cleveland, Ohio; and Herman K. Goldberg Fund, Baltimore, Md; unrestricted research grant from Research to Prevent Blindness, Inc, New York, NY; the Guerrieri Fund, Baltimore, Md; core grant 5P30EY01765-21 from the National Eye Institute, Bethesda, Md; and 1997 Association for Research in Vision and Ophthalmology/Retina Research Foundation/Joseph M. and Eula C. Lawrence Travel Fellowship Grant.Presented in part at the meetings of the American Academy of Ophthalmology, Chicago, Ill, October 1996, the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Fla, May 1997, and the Retina Society, Vancouver, British Columbia, September 1997.Corresponding author: Daniel Finkelstein, MD, Retinal Vascular Center, The Wilmer Ophthalmological Institute, The Johns Hopkins Medical Institutions, 600 N Wolfe St, Maumenee 219, Baltimore, MD 21287.

Wall, Michael; Johnson, Chris A.; Kutzko, Kim E.; Nguyen, Richard; Brito, Caridad; Keltner, John L.

1998 JAMA Ophthalmology

doi: 10.1001/archopht.116.1.53pmid: 9445208

ObjectiveTo measure the short- and long-term variability of automated perimetry in patients with optic neuritis and normal subjects.DesignProspective case-control design of patients with recovered optic neuritis with intraday and interday repetitions to obtain robust variability measurements. Entry criteria included a corrected pattern SD that was worse than the normal 5% probability level and a mean deviation worse than −3 dB but better than −20 dB. Five Humphrey 30-2 full threshold tests were administered during a 7-hour period (1 test every 2 hours) on the same day and at the same periods on 5 separate days.SubjectsSeventeen patients with recovered optic neuritis and 10 healthy subjects of similar age.Main Outcome MeasuresShort-term variability and long-term variability for global visual field data.ResultsPatients with optic neuritis demonstrated variations in visual field sensitivity that were outside the entire range of variability for normal controls. These variations occurred for multiple tests performed on the same day at specific times and for tests performed at specific times on different days. There were no consistent patterns of sensitivity changes that could be attributed to time of day. The most dramatic fluctuations occurred in a patient whose visual fields varied from normal to a hemianopic defect from one week to another and from a partial quadrant loss to a hemianopic defect at different times on the same day. Seven of the patients with optic neuritis also demonstrated intermittent vertical step defects.ConclusionsPatients with resolved optic neuritis can have large variations in visual field results on different days and at different times on the same day. The variations affect both the severity and the pattern of visual field loss and do not appear to be consistent across patients. These data indicate that care must be taken when automated visual field results in patients with optic neuritis are interpreted. Distinguishing systematic changes in sensitivity from variability requires more than a comparison of the current visual field with the most recent previous visual field.THE ABILITY to distinguish visual field progression or improvement from one visit to the next is difficult because of the variability that occurs with threshold measurements, especially in areas of visual field damage. This variability of conventional automated perimetry has been investigated extensively in normal subjects and patients with glaucomaand is closely related to loss of sensitivity. Beyond approximately 1 log unit (10 dB) of sensitivity loss in patients with glaucoma, variability rises exponentially and encompasses nearly the full measurement range of the instruments used.For example, a test location with 15 dB of loss in patients with glaucoma has a 95% prediction interval that ranges from about 5 to 30 dB.Less is known about the variability in visual field sensitivity that occurs in optic neuritis and multiple sclerosis. In contrast to glaucomatous damage, optic neuritis is characterized by immune-mediated inflammatory damage to the optic nerve, with the myelin sheath being the primary target of the inflammation. The main difference between glaucomatous visual field damage and optic neuritis–associated visual field loss is that in optic neuritis, the nerve fiber bundles that subserve central vision (the papillomacular bundles) are involved as often as the paracentral superior and inferior arcuate nerve fiber bundles.Early visual field loss in glaucoma, on the other hand, is predominantly found in the superior and inferior arcuate nerve fiber bundle regions, and central visual loss typically does not occur until advanced stages of the disease process.In view of the differences between glaucoma and optic neuritis, it is not clear that the response variability found in glaucomatous visual fields can be generalized to visual field loss derived from optic neuritis or multiple sclerosis. Results from the Optic Neuritis Treatment Trialindicate that optic neuritis produces a variety of patterns and degrees of visual field loss, both among different individuals and in the same individual at different times during the disease process. Although this qualitative information is helpful, it is also important to quantitatively document these variability characteristics to provide an empirical basis for evaluating whether visual fields in these patients are improving, getting worse, or simply demonstrating fluctuations. The purpose of the present study was to perform a formal investigation of the short-term and long-term variability of automated perimetry threshold determinations in patients with residual visual field loss caused by optic neuritis and/or multiple sclerosis in comparison with findings in healthy control subjects of similar age.SUBJECTS AND METHODSSUBJECTSSeventeen patients with a clinical history of optic neuritis and/or multiple sclerosis and 10 healthy subjects of similar age participated in the study. Before testing, informed consent was obtained from each participant according to the tenets of the Declaration of Helsinki. The study protocol was approved by the institutional review boards of both the University of Iowa, Iowa City, and the University of California, Davis.The control subjects were paid volunteers who were hospital employees or students. We selected them so that their ages would fall within the expected age range of patients with optic neuritis (20-50 years). Five healthy subjects were enrolled at each institution. These subjects were included if they had no history of eye disease or surgery, had no more than 5 diopters (D) of spherical equivalent and 3 D of astigmatic refractive error, and had normal results of ophthalmologic examination and automated perimetry by means of the Humphrey Field Analyzer (Humphrey Instruments, San Leandro, Calif) program 30-2. Potential controls were excluded if they had visual field indexes (mean deviation [MD], pattern SD [PSD], short-term fluctuation [SF], or corrected pattern SD [CPSD]) that were at the P<.05 probability level or worse, had 3 or more adjacent test locations with a total deviation score at the P<.05 probability level or worse, or had 2 or more adjacent test points with a total deviation score at the P<.01 probability level or worse. One eye was randomly selected for testing in the normal control subjects.Patients with optic neuritis were recruited from Optic Neuritis Treatment Trial patients (Iowa), by telephone calls after a patient database search (Iowa and Davis), and by referral from local multiple sclerosis societies (Davis). Ten patients with optic neuritis were enrolled at the University of California, Davis, and 7 were enrolled at the University of Iowa. All of the patients with optic neuritis had a clinical history of optic neuritis with residual visual field loss. Nine of the 10 Davis patients and 5 of the 7 Iowa patients also had multiple sclerosis. To be eligible, patients with optic neuritis had to have at least 2 previous visual fields in which the CPSD was outside the P<.05 normal probability level and an MD that was worse than −3 dB but better than −20 dB in one or both eyes. If none of the previous visual fields had been conducted within the past 6 months, an additional eligibility visual field was performed. In the event that both eyes qualified for the study, one was randomly selected for testing. The patients underwent a complete neuro-ophthalmologic examination at theinitial visit and were excluded if they had any ocular or neurologic disorders other than optic neuritis and/or multiple sclerosis or if they had refractive errors greater than 5 D spherical equivalent or 3 D astigmatic error. One patient only underwent the multiple test sessions on the same day, and 1 patient only participated in the multiple test sessions on different days. Therefore, comparisons within the 2 testing intervals were performed on the data from 16 patients, and comparisons between the 2 testing intervals (same day compared with weekly) were conducted on the data from 15 patients.PROCEDUREAfter the qualifying examinations, all participants underwent perimetric testing with the Humphrey Field Analyzer 30-2 full threshold program on one eye, according to the schedule shown in Table 1. Ten perimetric sessions were conducted, 5 at intervals of 1 to 2 hours on the same day and 5 with testing at various times of the day and separated by a 1-week interval between test sessions. The standard test conditions for the Humphrey Field Analyzer, 31.5-apostilb background, Goldmann size III target, and 200-millisecond stimulus duration, were used for all perimetric examinations. An appropriate near correction for the test distance was used for the tested eye, and an eye patch was used to occlude the nontested eye. During each examination, rest breaks were given when requested by the participant.Table 1. Testing ScheduleSee table graphicSTATISTICAL ANALYSISThe visual field data from all participants were imported into the SAS (Cary, NC) and Sigmastat (San Rafael, Calif) statistical analysis packages. The primary outcome variables were all normally distributed according to the Kolmogorov-Smirnov test (P>.05). All had similar variances by means of the Levene median test (P>.05). Analysis of variance (ANOVA) with post hoc ttests corrected for multiple comparisons were used for all statistical evaluations except those that failed the above tests for normality and homoscedasticity, in which case we performed an ANOVA on ranks with post hoc tests by the Student Neuman-Keuls method. The P<.05 probability level was used as the criterion for statistical significance.Differences in thresholds between groups and at different times of the day were tested for statistical significance by means of repeated-measures ANOVA with a 3-factor nested design. The between-subjects factor was group (healthy subjects and patients with optic neuritis) and the within-subjects factors were interval (same day and different days) and time (hour of the day). The dependent measures were mean threshold, foveal threshold, MD, SF, PSD, and CPSD. An ANOVA was also used to test for differences in variabilities for the same dependent measures. Interval was the within-subject variable and group was the between-subject variable.Differences in variability (SDs of the 5 tests) between intervals (all tests in 1 day vs different days) were tested for statistical significance by means of a general linear models procedure for a 2-factor nested design. The between-subjects factor was group (healthy subjects and patients with optic neuritis) and the within-subjects factor was interval (same day and different days). Subjects were nested within groups.We developed software with the use of Visual Basic in Microsoft Excel (Microsoft Corp, Redmond, Wash) to perform simulations of automated visual fields. To estimate the slope (SD of the cumulative gaussian function) of the frequency of seeing function for each possible visual sensitivity of the dynamic range of the Humphrey Field Analyzer, we used results from frequency of seeing experiments in patients with glaucoma.We evaluated the relationship between the slope of the frequency of seeing function for these data by finding the best fit of various simple functions. A power function with an rof 0.84 was used. Using this function, we were able to calculate a slope of the frequency of seeing function for the visual sensitivity for each test location. By then specifying the false-positive rate (5%) and false-negative rate (1%) for each test location, we could estimate the probability that a subject would respond to a stimulus at each possible sensitivity of the instrument we were simulating. Then, using (1) a 4/2 staircase procedure and (2) the starting values (25 dB) for the primary or "seed" points, (3) taking the mean of the doubly determined primary point, and (4) using the rules for passing other starting values within each quadrant (once a threshold was found, eccentricity-corrected values were passed to all adjacent locations not yet tested), we were able to simulate a standard algorithm Humphrey visual field examination. We assumed that any variability we found could be attributed mostly to the testing method rather than patient factors.RESULTSThe mean age of the patients was 41.7±9.9 years; the mean age of the controls was 34.6±7.5 years. The age difference between the 2 groups was not statistically significant. As indicated in Table 2, the patients with optic neuritis had significantly greater elapsed test times, questions asked, false-negative trials, false-negative errors, and false-positive trials than the controls. There was no significant difference in the number of false-positive errors between the 2 groups.Table 2. Basic Test StatisticsSee table graphicThe MD, PSD, SF, and CPSD in the patients with optic neuritis were all significantly different from the results of the controls (Table 3). The mean within-subject SDs of MD for the 5 tests in control subjects was 0.46 dB for the same-day measures and 0.36 dB for weekly visual field measures. In the patients with optic neuritis, the SDs of MD for the 5 same-day measures was 1.35 dB and for the different-day measures it was 3.10 dB. This represents a 3-fold increase in variability for the patients with optic neuritis in comparison with the control subjects for the same-day measures and a 9-fold increase in variability for the different-day measures. These differences were even more pronounced when the visual field quadrants were analyzed in a similar manner (Table 4).Table 3. Test IndexesSee table graphicTable 4. Test Results for the Quadrants and Fovea*See table graphicFigure 1presents the MD for all 5 visual field determinations for control subjects and patients with optic neuritis for same-day and weekly determinations. Note that the normal MD values are all tightly clustered, whereas the results for the patients with optic neuritis are more dispersed. In addition, it appears that there are considerable individual differences in variability of MD values among patients with optic neuritis. Similar results for PSD, SF, and CPSD are presented in Figure 2, Figure 3, and Figure 4with a similar format.Figure 1.Mean deviation values for healthy subjects and patients with optic neuritis or optic neuritis and multiple sclerosis (ON/MS) on the same day (A) and on different days (B). Note the large variability in the patients.Figure 2.Pattern SD values for healthy subjects and patients with optic neuritis or optic neuritis and multiple sclerosis (ON/MS) on the same day (A) and different days (B). Note the large variability in the patients.Figure 3.Short-term fluctuation values for healthy subjects and patients with optic neuritis or optic neuritis and multiple sclerosis (ON/MS) on the same day (A) and different days (B). Note the large variability in the patients.Figure 4.Corrected pattern SD for healthy subjects and patients with optic neuritis or optic neurtitis and multiple sclerosis (ON/MS) on the same day (A) and different days (B). Note the large variability in the patients.To evaluate the effects of group and test interval on threshold, the repeated-measures ANOVA with the 3-factor nested design showed that each of the dependent measures was significantly different between the control and patient groups (mean threshold, P<.001; foveal threshold, P<.001; MD, P<.001; PSD, P<.001; SF, P<.001; CPSD, P<.001). For MD, there was an interaction between group and test interval; there was no significant difference between same-day and different-day measures in control subjects, whereas there was a significant difference between same-day and different-day measures for the patients with optic neuritis. For the total average score, there was a significantly (P=.05) higher score (better sensitivity) of 1.36 dB for the different-day measures as compared with the same-day measures (includes both patients and normal subjects). There was no significant effect of time of day.The ANOVA for the SDs of the 5 visual field examinations by time interval disclosed significant differences among the dependent measures (mean threshold, P=.02; foveal threshold, P=.04; MD, P=.02; PSD, P=.01; SF, P=.009; CPSD, P=.02). There were significant differences between the groups but not between the testing intervals (control subjects and patients combined). However, when threshold was entered as a cofactor for the average total score, there were no significant differences between the groups of subjects for these dependent measures. Note in Figures 1 through 4 that there appears to be more variability in the visual field measurements from week to week than from within a day.Individual examples of visual fields obtained on the same day and weekly are presented in Figure 5. Figure 5, A, shows the results of a typical set of visual field determinations for a control subject. Note that there is minimal variation in the pattern of visual field sensitivity for the 10 test procedures. Figure 5, B, shows visual field loss of a patient with optic neuritis in the inferior nasal quadrant of the right eye. Both the severity and pattern of visual field loss appear to be consistent, both for visual field measures obtained on the same day and for those obtained on different days separated by 1-week intervals. About one fifth of the patients with optic neuritis demonstrated this degree of consistency among multiple visual field tests; about 30% showed moderate amounts of variability. However, approximately half of the patients with optic neuritis exhibited large variations in visual field results from one test to the next.Figure 5.Gray-scale and probability results in healthy subjects and patients with optic neuritis. Results are shown for the same day and different days matched for time of day. The 2 fields at the top of each graph are from 8 AM, the next pair down from 10 AM, etc. A, Typical example of a normal subject's consistent results. B, A patient with optic neuritis with consistent results. C, A patient with optic neuritis with variable results. Note the variation from near normal to near complete hemianopia. D, Another patient with variable results.Two dramatic examples of this high variability are shown in Figure 5, C and D. Figure 5, C, shows that for the same-day measures, this patient had a small superonasal paracentral visual field deficit for the first visual field performed in the morning. The results became progressively worse as the day progressed, resulting in a hemianopic defect by the late afternoon. The measures obtained on different days, separated by a week, range from a nearly normal visual field in the first week to a hemianopic defect in the third week to a small superonasal hemianopic deficit in the fifth week. A similar type of variation in both the pattern and severity of visual field loss is shown in Figure 5, D, for another patient.COMMENTVariability of visual field measurements in recovered patients with optic neuritis with −3 to −20 dB of residual visual field sensitivity loss was significantly higher than in healthy subjects of similar age, both for short-term (same day) and long-term (weekly) comparison intervals. We observed 3 patterns of variability among the patients with optic neuritis. Approximately 20% of the patients had good reproducibility for multiple visual field examinations, being equivalent to or only slightly worse than the variability demonstrated by control subjects. Moderate variability of visual field results was observed in approximately 30% of the patients with optic neuritis. About half of these patients demonstrated large variability in visual field measurements, in some instances ranging from normal or minimally abnormal visual fields to dense hemianopic deficits over a week or two or even within the same day. Similar patterns of variability were observed for PSD, CPSD, SF, and reliability indexes (with the exception of false-positive responses). These findings indicate that the response properties of the majority of patients with optic neuritis undergoing visual field testing exhibit significant fluctuations.Burde and Gallinreported elevated thresholds in patients with resolved optic neuritis by means of static perimetry, even though kinetic perimetry results yielded normal findings. This variation in sensitivity between static and kinetic perimetry results in optic neuritis, sometimes referred to as "statokinetic dissociation," has subsequently been reported by other investigators.Harmsin 1976 described the generally high variability among repeated visual field tests in patients with optic neuritis, and the first systematic study of the variability of visual thresholds in patients with multiple sclerosis was conducted by Patterson and colleagues in 1980.A number of factors, including temperature, fatigue, attention, time of day, axonal damage, "cross talk" among nerve fibers, extent of visual damage, and other related determinants, have been proposed to account for this high variation in visual field sensitivity. In the present study, we found no evidence that fatigue or time of day was strongly related to the variability of visual field measurements in patients with optic neuritis. As reported in glaucoma,we observed a strong direct relationship of threshold and variability.The magnitude of our long- and short-term variability is somewhat higher than that found in glaucoma (Table 5). Variability might be inherently higher in optic neuritis because of myelin loss surrounding surviving axons and related neuronal impulse slowing and instability. Alternatively, the variability of optic neuritis in this study may appear higher because our sample size is smaller than those in the published glaucoma studies, and our selection criteria excluded patients with mild defects (<3 dB of MD loss). Variability measures might also be different because the studies cited in Table 5were performed on the Octopus (Interzeag, Schlieren, Switzerland) rather than the Humphrey perimeter. As discussed below, some of the visual field variability is likely caused by an interaction between the shallow slopes of frequency of seeing curves at damaged test locations and the method of passing starting values within each quadrant as a test progresses. Early Octopus perimeters used as starting values the first visual field of normal values corrected for age. Subsequent fields used values from a "master field" that was a composite of previous thresholds from that patient. Later Octopus perimeters have adopted the 4-quadrant primary point to adjacent test location strategy (see below).Table 5. Long-term and Short-term Variability of Optic Neuritis and Glaucoma*See table graphicAbout 40% of the patients with optic neuritis demonstrated visual field deficits that appeared to have a vertical step component (usually in the form of a quadrant defect). None of the control subjects had a step defect. None of the patients with vertical step defects had a homonomous defect in the fellow eye, and the defects respected the horizontal meridian in all of the patients but 2. Many of these vertical step deficits were present intermittently over repeated test sessions, and their presence was more frequently observed in those patients with greater amounts of variability. Seven of the weekly determinations and 3 of the same-day sessions exhibited vertical step deficits that were intermittent. Although some of the vertical steps may be related to anatomical or pathophysiological factors in these patients, we believe that many of these vertical step patterns in this patient population may be caused by the testing algorithm used by the Humphrey Field Analyzer (and the Octopus perimeter).With the full-threshold strategy of the Humphrey Field Analyzer, "primary points" (9° vertical and 9° horizontal displacement from fixation) are the first to be determined in each visual field quadrant. These primary point thresholds are doubly determined and the mean is calculated. The primary point threshold is then used as a basis (with a correction for eccentricity) for the starting value for each of the 8 neighboring points surrounding the primary point. In turn, the thresholds of these secondary points are used as the basis for starting values of adjacent test locations within the quadrant. That is, the passing of starting values does not cross the horizontal or vertical meridians. Because the results of staircase procedures can be influenced by the starting position of the staircase (especially when variability and response errors are high), a low threshold for a primary point could make it more likely that the entire quadrant might produce lower sensitivity values and the appearance of a vertical step or quadrant defect.To test this possibility, we performed 2 analyses. The first consisted of comparing the mean of the primary point sensitivities in a particular quadrant (eg, 9° up and 9° right from fixation) to thresholds from 6 equidistant test locations within the same quadrant displaced vertically (eg, 3° up/21° right, 9° up/21° right, and 15° up/21° right) and horizontally (eg, 21° up/3° right, 21°up/9° right, and 21° up/21° right). A correlation coefficient was then computed for the primary point sensitivity and the mean of these 6 within-quadrant points. This was compared with 6 other locations that were outside of the quadrant (thereby influenced by a different primary point) but were the same distance from the primary point. These test sites, outside the quadrant, were the mirror image of the 6 test locations within the same quadrant. Our hypothesis was that if the starting value of the staircase was a significant factor in the sensitivity measures of the quadrant, because of a combination of the primary point sensitivity and high response variability, then the within-quadrant points (directly influenced by the primary point) would be more highly correlated with the primary point sensitivity than the equidistant outside-quadrant points (predominantly influenced by a different primary point). If this relationship is true, it may account for the presence of some vertical step (quadrant) defects. We suspect this mechanism also applies to step defects in other patients with visual field damage, including patients with glaucoma. Future observations should clarify the generality of this mechanism.Results of this analysis are presented in Table 6. They show that the within-quadrant sensitivity values are more highly correlated with the primary point sensitivity than those equidistant test locations outside the quadrant. For the within-quadrant points, the rwas 0.55 for same-day measurements and 0.50 for weekly measurements. In comparison, the outside-quadrant points had an rof 0.19 for the same-day measures and 0.16 for the weekly measurements. These differences were statistically significant at the P=.009 level (paired ttest).Table 6. Correlation Coefficients for the Quadrant Primary Point and Equidistant Test Locations Inside and Outside the QuadrantSee table graphicThe second analysis consisted of simulating visual field tests with conditions of the primary point having a moderately low sensitivity value. We chose visual field data from one perimetric examination from a patient with high variability and used these results as the "true" visual sensitivities used to calculate frequency of seeing curve slopes for each test location (see Figure 5, D, fourth field of different days; and Figure 6, A). We then simulated 30 visual fields by means of the Humphrey perimetry methods of program 30-2. Figure 6, B, shows the field with the lowest right-upper-quadrant thresholds; Figure 6, A, shows the field with the highest right-upper-quadrant thresholds. All of the simulated fields showed defects that were less dense (higher sensitivities) than the actual values. This is probably because of the right-upper-quadrant primary point of 18 dB and values being passed to the surrounding points that are more than 1 log unit higher than the "true" thresholds. In Figure 6, it can be seen that these simulated conditions can produce the appearance of intermittent quadrant and vertical steplike deficits. In view of both of these results, we believe that at least some of the transient vertical steps that were present in the visual fields of patients with optic neuritis in our investigation were the result of an interaction between the Humphrey Field Analyzer's testing algorithm and the high response variability (related to the shallow slope of the frequency of seeing curve in damaged test locations).Figure 6.Results of visual field simulation of 30 visual fields by means of thresholds from a patient with optic neuritis who had variable results of conventional automated perimetry strategy. A, Actual values. B and C, Visual field with the highest (B) and lowest (C) right-upper-quadrant sensitivity. Note how the appearance of a quadrant defect can be simulated.In summary, we found large variations in visual field measurements obtained in many patients with recovered optic neuritis who had residual visual field loss of −3 to −20 dB. Our study suggests that some of these large changes in the visual field are likely related to the testing method rather than to new disease activity. Therefore, treatment decisions that are based solely on a comparison of the current visual field examination with the most recent previous visual field should be avoided. We hope that the use of larger test stimuli,more robust test strategies, and other refinements of perimetry in the future will help to reduce the difficulties associated with high variability in the visual fields of patients with optic neuritis. Until that time, caution in the interpretation of test results, multiple retests of the visual field, and examination of long-term trends over many visual field examinations should be exercised in any treatment decisions concerning patients with optic neuritis that are based on perimetric test results.RJBoeglinJCaprioliMZulaufLong-term fluctuation of the visual field in glaucoma.Am J Ophthalmol.1992;113:396-400.JFlammerSMDranceFFankhauserLAugustinyDifferential light threshold in automated static perimetry: factors influencing short-term fluctuation.Arch Ophthalmol.1984;102:876-879.EBWernerNSahebDThomasVariability of static visual threshold responses in patients with elevated IOPs.Arch Ophthalmol.1982;100:1627-1631.EBWernerBPetrigTKrupinKIBishopVariability of automated visual fields in clinically stable glaucoma patients.Invest Ophthalmol Vis Sci.1989;30:1083-1089.JFlammerFluctuations in the visual field.In: Drance SM, Anderson DR, eds. Automatic Perimetry in Glaucoma: A Practical Guide.Orlando, Fla: Grune & Stratton; 1985:161-173.JFlammerSMDranceMZulaufDifferential light threshold: short- and long-term fluctuation in patients with glaucoma, normal controls, and patients with suspected glaucoma.Arch Ophthalmol.1984;102:704-706.AHeijlALindgrenGLindgrenTest-retest variability in glaucomatous visual fields.Am J Ophthalmol.1989;108:130-135.JLKeltnerCAJohnsonJOSpurrRWBeckBaseline visual field profile of optic neuritis: the experience of the optic neuritis treatment trial: Optic Neuritis Study Group.Arch Ophthalmol.1993;111:231-234.MWallRJMawKEStanekBCChauhanThe psychometric function and reaction times of automated perimetry in normal and abnormal areas of the visual field in glaucoma patients.Invest Ophthalmol Vis Sci.1996;37:878-885.MWallKSKutzkoBCChauhanVariability in patients with glaucomatous optic nerve damage is reduced using size V stimuli.Invest Ophthalmol Vis Sci.1997;38:426-435.RMBurdePFGallinVisual parameters associated with recovered retrobulbar optic neuritis.Am J Ophthalmol.1975;79:1034-1037.CHudsonJMWildAssessment of physiologic statokinetic dissociation by automated perimetry.Invest Ophthalmol Vis Sci.1992;33:3162-3168.LWedemeyerJLKeltnerCAJohnsonStatokinetic dissociation in optic nerve disease.Doc Ophthalmol Proc Ser.1989;43:9-14.HHarmsRole of perimetry in assessment of optic nerve dysfunction.Trans Ophthalmol Soc U K.1976;96:363-367.VHPattersonDHFosterJRHeronVariability of visual threshold in multiple sclerosis: effect of background luminance on frequency of seeing.Brain.1980;103:139-147.MZulaufJCaprioliDCHoffmanCSTresslerFluctuation of differential light sensitivity in clinically stable glaucoma patients.In: Mills RP, Heijl A, eds. Perimetry Update 1990/1991: Proceedings of the Xth International Perimetric Society Meeting.Amstelveen, the Netherlands: Kugler Publications; 1991:183-188.EBWernerGGanibanAGBalazsiEffect of test point location on the magnitude of threshold fluctuation in glaucoma patients undergoing automated perimetry.In: Mills RP, Heijl A, eds. Perimetry Update 1990/1991: Proceedings of the Xth International Perimetric Society Meeting.Amstelveen, the Netherlands: Kugler Publications;1991:175-181.Accepted for publication August 29, 1997.This study was supported in part by a Veterans Affairs Merit Review Grant (Dr Wall); research grants EY-03424 (Dr Johnson) and EY-09435 (Dr Keltner) from the National Eye Institute, Bethesda, Md; unrestricted research grants from Research to Prevent Blindness Inc, New York, NY (to the Departments of Ophthalmology at University of Iowa and University of California, Davis); and cooperative agreement EY09435 (Roy W. Beck Jaeb Center for Health Research, Tampa, Fla) from the National Eye Institute.We thank our patients for their cooperation and for tolerating the rigors of the automated perimetry testing protocol used in this study. We also thank Kimberly Cello and Jacqueline Nelson-Quigg for their assistance in collecting visual field data.Reprints: Michael Wall, MD, Department of Neurology, University of Iowa College of Medicine, 200 Hawkins Dr, #2007 RCP, Iowa City, IA 52242-1053 (e-mail: [email protected]).

Sawaguchi, Shoichi; Fukuchi, Takeo; Abe, Haruki; Kaiya, Tadayoshi; Sugar, Joel; Yue, Beatrice Y. J. T.

1998 JAMA Ophthalmology

doi: 10.1001/archopht.116.1.62pmid: 9445209

ObjectiveTo examine the 3-dimensional collagen fibril organization in the Bowman layer of keratoconus corneas.MethodsEight keratoconus corneas, 8 corneas with other diseases, and 5 normal human corneas were studied. A cell maceration method in combination with scanning electron microscopy was used to examine the collagen network in the Bowman layer.ResultsIn normal corneas, the surface of the Bowman layer was smooth and collagen fibrils were regularly arranged. By contrast, sharply edged defects in the Bowman layer were found in keratoconus corneas. Lattice-like configurations of the ruptured Bowman layer and collagenous scar tissue were observed, to varying degrees, in all keratoconus corneas examined. None of the other diseased corneas exhibited the ruptures.ConclusionsScanning electron microscopy demonstrated alterations in the Bowman layer specific to keratoconus. Fragmentation of the Bowman layer may be an early change leading to keratoconus conditions.KERATOCONUS is a noninflammatory disease characterized by thinning and scarring of the central portion of the cornea.Histopathologic and ultrastructural studies have demonstrated that in early stages of the disease, fragmentation of the epithelial basement membrane occurs with disintegration of the Bowman layer and fibrillation of the anterior stroma.The central cornea then becomes thinned, with destruction of the Bowman layer and stromal scarring. A loss of collagen lamellae occurs, but the collagen fibrils are of normal diameter.The lamellae are surrounded by granular materials, which are shown to be rich in neutral polysaccharides and glycoproteins.In advanced stages of the disease, the central portion of the Descemet membrane may rupture, resulting in acute hydrops.Electron microscopic studies aimed at 2-dimensional examination of keratoconus corneas were performed more than 20 years ago. Since then, the technology, resolution, and machinery have vastly improved.Our investigation sought to revisit the structure of keratoconus corneas, focusing particularly on the 3-dimensional collagen architecture in the Bowman layer. Direct observation of this structure by conventional techniques has been infeasible because of the overlying corneal epithelium. With a recently developed cell maceration and tissue conductive method,we removed the corneal epithelium and visualized the 3-dimensional collagenous architecture of the Bowman layer under scanning electron microscopy. Multiple ruptures in the Bowman layer of keratoconus corneas and the possible sequence of events were demonstrated.MATERIALS AND METHODSTISSUESEight corneal buttons from patients with typical clinical features of keratoconus (Table 1) were obtained at the time of penetrating keratoplasty from the Cornea Service of University of Illinois at Chicago College of Medicine or Seirei Hamamatsu Hospital, Shizuoka, Japan. Patients ranged in age from 18 to 46 years at the time of surgery. Five normal human corneas from donors (aged 2, 19, 43, 53, and 66 years) were obtained from the Illinois Eye Bank, Chicago, within 24 hours of death. The corneas were clear and the donors did not have known ocular disease.Clinical Characteristics of Patients With KeratoconusSee table graphicTo serve as another set of controls, corneas were obtained from 8 patients with other corneal diseases. They were from 2 patients (aged 69 and 70 years) with pseudophakic bullous keratopathy, 1 patient (aged 18 years) with bullous keratopathy associated with absolute glaucoma, 1 patient (aged 55 years) with corneal scar after ulceration, 2 patients (aged 55 and 58 years) with lattice corneal dystrophy, 1 patient (aged 48 years) with granular corneal dystrophy, and 1 patient (aged 51 years) with herpetic interstitial keratitis.SCANNING AND TRANSMISSION ELECTRON MICROSCOPYCorneas obtained were fixed immediately in 2.5% glutaraldehyde and 2% formalin in Sorensen phosphate buffer solution for 2 to 5 days and transferred to buffered formalin until processing.The cell maceration method was carried out as previously described.Briefly, the fixed specimens were rinsed in distilled water overnight, immersed at room temperature for 5 days in a 10% sodium hydroxide solution, and rinsed thoroughly with several changes of distilled water for 24 hours. They were then subjected to a conductive staining method by soaking in 2% tannic acid for 3 hours, washed in distilled water for 1 hour, and postfixed in aqueous 1% osmium tetroxide for 3 hours. After dehydration through a graded ethyl alcohol series, the specimens were transferred to isoamyl alcohol, critical-point dried, mounted on aluminum stabs, and coated with gold in an ion coater (Hitachi, Tokyo, Japan). Scanning electron microscopy was performed with a scanning electron microscope (Hitachi S-2300) at an accelerated voltage of 25 kV.To ensure that the corneal specimens were processed properly, pieces of the tissue were embedded in epoxy resin after cell maceration and conductive staining. Ultrathin sections were double stained with uranyl acetate–lead citrate and were observed under a transmission electron microscope (Hitachi H-7000).RESULTSTable 1summarizes the clinical features of 8 patients with keratoconus, including age at onset, age at surgery, and ocular manifestations. Patients 5 and 6 had a history of acute hydrops and patients 4 and 8 had relatively mild cases. None of the patients had a family history of the disease except for patient 7, whose brother had keratoconus. Routine pathologic examinations showed the typical features of keratoconus, including breaks in the Bowman layer, scarring, and iron ring.As evidenced by transmission electron microscopy, treatment of corneas with 10% sodium hydroxide solution removed most of the cellular elements, basal laminae, and other extracellular materials, leaving collagen fibrils relatively intact (Figure 1). The scanning electron micrographs shown in Figure 2depict the surface of the Bowman layer from normal human corneas at varying magnifications. At lower magnifications (Figure 2, A and B), smooth surface of the Bowman layer was observed. At higher magnifications (Figure 2, C and D), the Bowman layer displayed a honeycomblike porous structure, made up of a dense meshwork of collagen fibrils. Similar features were found in all 5 normal human corneas studied.Figure 1.Transmission electron micrographs of corneas from (A) a 19-year-old normal subject and (B) a 22-year-old keratoconus-affected patient (patient 1) after treatment with sodium hydroxide. Note that almost all cellular and extracellular elements appear to be removed and only collagen fibrils remain relatively intact. BM indicates Bowman layer; ST, corneal stroma. Arrowheads point to the edge of ruptured BM layer in keratoconus.Figure 2.Scanning electron microscopic observations of the Bowman layer from a 19-year-old normal subject at varying magnifications. At lower magnifications (A and B), the surface of Bowman layer appears to be slightly wavy. At higher magnifications (C and D), the regular collagen fibrillar network displays a honeycomb pattern.In contrast, multiple sharply edged defects in the Bowman layer (Figure 3) were noted in all the keratoconus corneas examined. A lattice-like configuration of ruptured Bowman layer was found. Hypertrophic collagenous proliferation, which partially or totally replenished the ruptures, was observed. The fact that patients 5 and 6 (Figure 3, D and E) had a history of acute hydrops may explain why much more collagenous proliferation was observed.Figure 3.Scanning electron microscopic findings in corneas from 7 patients with keratoconus (A through G, patients 2 through 8). Specimens show varying degrees of sharply edged ruptures in the Bowman layer. Hypertrophic collagenous scar tissue fills the gaps. More collagenous scar tissue covering the Bowman layer (asterisks) is found in specimens from patients 5 (D) and 6 (E), who had a clinical history of acute hydrops.Under a higher magnification, the proliferation of collagenous tissue and ruptures in the Bowman layer could be viewed more clearly (Figure 4, A). The ruptured areas of the Bowman layer were filled by proliferated collagenous tissue that appeared to derive from the anterior stroma just beneath the Bowman defects (Figure 4, B).Figure 4.Scanning electron micrographs of the cornea from a patient with keratoconus (patient 1). A, Proliferated collagenous tissue (arrows) and relatively normal area of the Bowman layer (lower left) are demonstrated. Between the 2 areas, defects of the Bowman layer can be seen (asterisks). B, Area between the asterisks in A at higher magnification. Sharply edged ruptures (arrowheads) can be seen. Defects of the Bowman layer appear to be occluded by proliferated collagenous tissue of anterior corneal stroma (arrows). Note that the honeycomb pattern on the surface of the Bowman layer is distorted around the gaps.When specimens were observed from a lateral view, normal human corneas (Figure 5, A) showed well-packed collagenous lamellae with spindle-shaped remains of sodium hydroxide–digested keratocytes and the Bowman layer (Figure 5, A, between white arrows) uniformly covering the stroma. In keratoconus corneas, misaligned Bowman layer (Figure 5, B, arrow), irregularly thinned Bowman layer (Figure 5, C) and defects (Figure 5, D) were demonstrated. With increasing severity of damage in the Bowman layer, the arrangement of collagenous lamellae also seemed to be more distorted into the deeper stroma (Figure 5, B through D) of keratoconus corneas. Under a higher magnification, the well-packed and well-aligned collagenous lamellae observed in normal human corneas (Figure 5, E) were replaced by the loose and randomly oriented collagen fibrils in keratoconus corneas (Figure 5, F). Multiple pores (Figure 5, F, asterisks) were seen.Figure 5.Lateral views of scanning electron microscopic findings from corneas obtained from a 43-year-old normal subject (A and E) and a patient with keratoconus (patient 1) (B through D and F). Compared with the regular Bowman layer (A), the keratoconus cornea showed irregularly thinned and partially disrupted Bowman layer (B through D). In the normal human cornea (A and E) and in areas at perhaps a very early stage of Bowman layer changes in keratoconus (B), collagen lamellae appear to be well packed and regularly organized. Only a few spindle-shaped defects are seen in B, compared with the increasingly distorted, loosely packed collagen lamellae with numerous irregular pores in moderate (C) to advanced (D and F) areas of the keratoconus specimen. Arrow in part B indicates the very early and mild changes of the Bowman layer in keratoconus; asterisks in part F, irregularly shaped stromal defects with irregularly and loosely arranged collagen fibrils in keratoconus.None of the other diseased corneas (Figure 6) exhibited these alterations in the Bowman layer under scanning electron microscopy. No multiple breaks were observed. The surface appearance was unlike that of the keratoconus cases even when prominent collagenous scar tissue could be seen, such as those in the cases of bullous keratopathy (Figure 6, A through C) and corneal scarring (Figure 6, D). Corneas from the patient with lattice corneal dystrophy (Figure 6, E) and the patient with granular corneal dystrophy (Figure 6, F) had a characteristic lattice or granular appearance, respectively.Figure 6.Surface views under scanning electron microscopy of other diseased corneas. Corneas were obtained from 70-year-old (A), 69-year-old (B), and 18-year-old (C) patients with bullous keratopathy, a 55-year-old patient with corneal scar (D), a 55-year-old patient with lattice corneal dystrophy (E), a 48-year-old patient with granular corneal dystrophy (F), and a 55-year-old patient with herpetic infection (G). Note that no findings such as multiple breaks of the Bowman layer seen in keratoconus (Figure 3) can be demonstrated. However, depending on disease conditions, different degrees of collagenous scar tissues are observed.COMMENTThe current scanning electron microscopic study after a cell maceration and tissue conductive procedure distinctly demonstrated the 3-dimensional organization of collagen fibrils in the Bowman layer of normal human, keratoconus, and other diseased corneas. This method, previously used successfully for examination of peripheral nerve,pial septa,cornea,sclera,and the Bruch membrane,allows a direct visualization of the architecture of collagen fibrils, which, to our knowledge, had not previously been attainable for the Bowman layer.Keratoconus specimens in this study, depending on disease severity, showed varying degrees of lattice-like, sharply edged ruptures and fragmentation of the Bowman layer. In more advanced areas, hypertrophic collagenous proliferation partially or totally filled the ruptures. The observed characteristic breaks and changes in the Bowman layer confirm and expand findings reported previously by light and electron microscopy.When the changes in the Bowman layer were minimal to mild, the corneal stroma underlying the Bowman layer correspondingly showed only minimal changes. However, the corneal stromal alteration advanced when damage in the Bowman layer became more evident in keratoconus.In 2 earlier light and electron microscopic studies, Chi et aland Tengpostulated that the ruptures of the Bowman layer were most likely to be replaced by tissue from stroma underneath the Bowman layer. Such a possibility was substantiated by this study. The ruptured areas appeared to be filled with proliferated collagenous tissue from the anterior stroma just beneath the ruptured Bowman layer.By slitlamp microscopy, anterior clear spaces have been observed within the thin portion of the conical protrusion in both early and advanced cases of keratoconus.Histologic examinations further revealed that the clinical finding correlated with breaks in the Bowman layer. It was suggested that the clear zones observed may represent breaks in the Bowman layer before the scar formation. In the current study, we are unable to determine whether our scanning electron microscopic findings can be correlated with the clinical finding, as slitlamp photographs were unavailable.Weand other investigatorshave reported that the collagen content in some cases of keratoconus is reduced compared with normal human corneas. In this study, loosely packed and randomly oriented collagen fibrils were demonstrated in some of the keratoconus corneal stroma, which may reflect the reduced collagen density. Consistent with the earlier histologic findings,abnormal chondroitin and dermatan sulfate–type proteoglycans were more recently found to accumulate in keratoconus corneas around collagen fibrils and collagen lamellae.The numerous pores noted in the stroma of keratoconus corneas in this study may thus represent areas occupied by keratocytes and by the abnormal proteoglycan molecules that are dislodged during the sodium hydroxide treatment. In nonkeratoconus diseased corneas, we noted no alterations in the Bowman layer characteristic of keratoconus changes.The Bowman layer is an acellular matrix at the interface between the corneal epithelium and the stroma. It links the epithelial basement membrane and the stroma proper and may be crucial for the epithelial attachment and function. During human corneal epithelial development, a distinct Bowman layer is formed at 19 weeks.After birth, the thickness of the Bowman layer remains unchanged. Components of Bowman layer are believed to be synthesized by both corneal epithelial and stromal cells and the epithelial-stromal interaction may be a major factor in the formation of the Bowman layer.Several years after radial keratotomy in human corneas, a Bowman layer–like structure was formed underneath epithelial plugs that extended into the stroma.The collagen fibrils in the Bowman layer are of relatively small diameter and are randomly arranged. It is unclear however how these collagens are organized or how they are maintained. In the underlying corneal stroma, the resident stromal cells are responsible for the maintenance and organization of the collagens. However, the Bowman layer is acellular. One possibility is that the maintenance is performed by the sparse stromal cells that transverse into the Bowman layer. Alternatively, cytokines may also be involved. In keratoconus, the maintenance function of the stromal cells or the cytokines may be disturbed both for the Bowman layer and for the stroma.The etiologic mechanism for the development of keratoconus is not fully clear. One hypothesis, based on data from our laboratoryand others,is that the degradation processes in keratoconus may be aberrant. Supporting evidence includes elevated lysosomal hydrolase activitiesand decreased levels of proteinase inhibitorsin keratoconus corneas. Through the course of enzyme and inhibitor studies, the corneal epithelium consistently shows the most dramatic biochemical abnormalities.It thus has been suggested that even though the thinning in keratoconus occurs primarily in the stroma, the corneal epithelium may also be involved in the disease development.The corneal epithelial theory was proposed based on an electron microscopic examination by Tengin the early 1960s. Lacking convincing evidence, however, this suggestion has since been considered as only conjecture.Our studiesshowing biochemical alterations mostly in the corneal epithelium provide more direct support for the theory. It is also corroborated by our finding that conjunctival epithelial cells from patients with keratoconus contain higher than normal lysosomal hydrolase activities.The sequence of events noted in this study further indicates that the changes in the Bowman layer precede those in the corneal stroma, suggesting that the corneal epithelium may be an important factor at the initial or early stage of keratoconus development. One scenario may be that the increased degradative enzymes and reduced inhibitors in the corneal epithelium trigger rupture and fragmentation of the Bowman layer. The subsequent environmental changes or the interactions between the epithelial cells and the genetically predisposed stromal cells may ultimately induce the thinning and scarring manifested in keratoconus.The epithelial-stromal interaction has been considered to be a factor involved in the development of keratoconus. Wilson et alhave postulated that interleukin 1 (IL-1) may be a modulator of epithelial and stromal interactions, regulating the corneal cell proliferation, differentiation, and death. They have also proposed a role of the IL-1 system in the causes of keratoconus. Interestingly, cultured keratoconus stromal cells have been shown to contain 4-fold higher binding sites for IL-1.An enhanced expression of IL-1 receptor has also been noted in keratoconus corneas.The IL-1 hypothesis is consistent with the degradation hypothesis because IL-1 is known to regulate the expression of matrix metalloproteinases in the cornea.JHKrachmerRSFederMWBelinKeratoconus and related non-inflammatory corneal disorders.Surv Ophthalmol.1984;28:293-322.HHChiBZKatzinCCTengHistopathology of keratoconus.Am J Ophthalmol.1956;42:847-860.CCTengElectron microscopic study of pathology of keratoconus.Am J Ophthalmol.1963;55:18-47.CCPataaLJoyonFRoucherUltrastructure of keratoconus.Arch Ophtalmol (Paris).1970;30:403-417.TIwamotoAGDevoeParticulate structures in keratoconus.Arch Ophtalmol (Paris).1975;35:65-72.AJBronKeratoconus.Cornea.1988;7:163-169.YPouliquenKeratoconus.Eye.1987;1:1-14.O0htaniTUshikiTTaguchiAKikutaCollagen fibrillar networks as skeletal frameworks: a demonstration by cell maceration/scanning electron microscopic method.Arch Histol Cytol.1988;51:249-261.TUshikiCIdeThree-dimensional organization of the collagen fibrils in the rat sciatic nerve as revealed by transmission and scanning electron microscopy.Cell Tissue Res.1990;260:175-184.SSawaguchiBYJTYueHAbeEIwataTFukuchiTKaiyaThe collagen fibrillar network in the human pial septa.Curr Eye Res.1994;13:819-824.YKomaiTUshikiThe three-dimensional organization of collagen fibrils in the human cornea and sclera.Invest Ophthalmol Vis Sci.1991;32:2244-2258.SSawaguchiTFukuchiMShirakashiHAbeTKaiyaMSawsThree dimensional architecture of Bowman's collagen fibrils in diseased corneas.Folia Ophthalmol Jpn.1995;46:1261-1285.SSawaguchiHAbeTFukuchiTKaiyaMSawsThree-dimensional scanning electron microscopic appearance of Bowman's condensation in keratoconus: a report of a case.Folia Ophthalmol Jpn.1996;47:50-52.TMatsuokaNMatsuoHNakagawaMDoteScanning electron microscopic observation of the collagen fibrillar network in Bruch's membrane by cell maceration.Nippon Ganka Gakkai Zasshi.1991;95:318-324.MBShapiroMMRodriguesMRMandelJHKrachmerAnterior clear spaces in keratoconus.Ophthalmology.1986;93:1316-1319.BYJTYueJSugarKBenvenisteHeterogeneity in keratoconus: possible biochemical basis.Proc Soc Exp Biol Med.1984;175:336-341.JWCritchfieldAJCalandraABNesburnMCKenneyKeratoconus, I: biochemical studies of normal and keratoconus corneas.Exp Eye Res.1988;46:953-963.TTAndreassenAHSimonsenHOxlundBiomechanical properties of keratoconus and normal corneas.Exp Eye Res.1980;31:435-441.SSawaguchiBYJTYueIChangJSugarJRobinProteoglycan molecule in keratoconus corneas.Invest Ophthalmol Vis Sci.1991;32:1846-1853.ASTisdaleSJSpurr-MichaudMRodriguesJHackettJKrachmerIKGipsonDevelopment of the anchoring structures of the epithelium in rabbit and human fetal corneas.Invest Ophthalmol Vis Sci.1988;29:727-736.EDHayDevelopment of the vertebrate cornea.Int Rev Cytol.1980;63:263-322.GRJMellesPSBinderMNMooreJAAndersonEpithelial-stromal interactions in human keratotomy wound healing.Arch Ophthalmol.1995;113:1124-1130.SSawaguchiBYJTYueJSugarJEGilboyLysosomal enzyme abnormalities in keratoconus.Arch Ophthalmol.1989;107:1507-1510.SSawaguchiSSTwiningBYJTYuePMWilsonJSugarS-KChanα1-Proteinase inhibitor levels in keratoconus.Exp Eye Res.1990;50:549-554.SSawaguchiSTwiningBYJTYueα2-Macroglobulin levels in normal human and keratoconus corneas.Invest Ophthalmol Vis Sci.1994;35:4008-4014.MEFiniBYJTYueJSugarCollagenolytic/gelatinolytic metalloproteinases in normal human and keratoconus corneas.Curr Eye Res.1992;11:849-862.DBrownMMChwaAOpbroekMCKenneyKeratoconus corneas: increased gelatinolytic activity appears after modification of inhibitors.Curr Eye Res.1993;12:571-581.TFukuchiBYJTYueJSugarSLamLysosomal enzyme activities in conjunctival tissues of patients with keratoconus.Arch Ophthalmol.1994;112:1368-1374.SEWilsonY-GHeJWengEpithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing.Exp Eye Res.1996;62:325-337.EJFabreJBureauYPouliquenGLoransBinding sites for human interleukin-1α, γ-interferon and tumor necrosis factor on cultured fibroblasts of normal cornea and keratoconus.Curr Eye Res.1991;10:585-592.LZhouRBWhitelockBYJTYueJSugarCytokine and receptor expression in keratoconus corneas.Invest Ophthalmol Vis Sci.1996;37(suppl):S1017.MTGirardMMatsubaraMFiniTransforming growth factor-β and interleukin-1 modulate metalloproteinase expression by corneal stromal cells.Invest Ophthalmol Vis Sci.1991;32:2441-2454.Accepted for publication July 25, 1997.This investigation was supported in part by research grants EY 03890 and EY 05628 and core grant EY 01792 from the National Eye Institute, National Institutes of Health, Bethesda, Md, and by the Research to Prevent Blindness (New York, NY) Senior Scientific Investigator Award (Dr Yue).Reprints: Beatrice Y. J. T. Yue, PhD, University of Illinois at Chicago College of Medicine, Department of Ophthalmology and Visual Sciences, 1855 W Taylor St, Chicago, IL 60612.

Yang, Chang-Sue; Khawly, Joseph A.; Hainsworth, Dean P.; Chen, San-Ni; Ashton, Paul; Guo, Hong; Jaffe, Glenn J.

1998 JAMA Ophthalmology

doi: 10.1001/archopht.116.1.69pmid: 9445210

ObjectiveTo determine the efficacy and pharmacokinetics of an intravitreal sustained-release triamcinolone acetonide and 5-fluorouracil (TA/5-FU) codrug in the treatment of experimental proliferative vitreoretinopathy (PVR).MethodsThe therapeutic efficacy of the TA/5-FU codrug was determined in a rabbit model that simulates human PVR. Intravitreal levels of triamcinolone and 5-fluorouracil were measured at different time points and drug release in vitro was tested. Toxic effects were evaluated by electroretinograpy, clinical examination, and light microscopy.ResultsBoth the severity of PVR grade and the percentage of eyes with moderate or worse tractional detachment were significantly less in eyes treated with the codrug. The therapeutic effect of the intravitreal codrug was paralleled by sustained intravitreal levels of triamcinolone and 5-fluorouracil. There were no drug-related toxic effects evident on clinical or histopathologic examination of eyes containing the TA/5-FU codrug.ConclusionsThe intravitreal sustained-release TA/5-FU codrug effectively inhibits the progression of PVR in a rabbit model that closely resembles PVR in humans. The TA/5-FU codrug may simultaneously target different components of the wound-healing response.PROLIFERATIVE vitreoretinopathy (PVR) refers to the migration and proliferation of cells into the subretinal space, vitreous cavity, and onto the retinal surface and undersurface. Subsequent collagen production and cell-mediated contraction of the collagenous scar leads to retinal detachment and loss of vision.Although refinements in surgical techniques and equipment have improved the success rate of surgery to repair retinal detachment in recent years, recurrence due to reproliferation is not uncommon and remains the leading cause of failure of retinal reattachment surgery.A major challenge in the successful long-term therapy of PVR is stabilization of anatomical reattachment through pharmacological manipulation of the wound-healing response. A variety of pharmacological adjuncts have been evaluated to reduce the proliferation of fibrous tissue within the eye both in animal models and in limited human trials.When delivered by intravitreal injection, the short half-life of these agents necessitates frequent injections, thereby limiting their clinical utility. When administered systemically, it is difficult to achieve adequate intraocular drug levels without producing systemic toxic effects. Proliferative vitreoretinopathy is limited to the eye, and reproliferation and recurrent retinal detachment occur within the first few months after the initial retinal reattachment surgery; thus, an intraocular drug delivery system that maintained therapeutic levels in the eye for several months would obviate the need for repeated intravitreal injections and would avoid toxic effects associated with systemic administration.Both triamcinolone acetonideand 5-fluorouracilindividually inhibit PVR in a rabbit model. We hypothesized that a device that contained both triamcinolone and 5-fluorouracil in combination may be more effective than treatment with either agent alone. In preliminary studies,we demonstrated the efficacy of a corticosteroid and 5-fluorouracil conjugate coated with polyvinyl alcohol polymer in the treatment of experimental PVR. The codrug represents a novel drug delivery system that is synthesized by linking a triamcinolone acetonide moiety to 5-fluorouracil via a modified labile carbonate bond. Once in the vitreous, dissolution of the conjugate followed by rapid hydrolysis allows both active components to be released in an equimolar ratio. Although the results were promising, there were several limitations of these initial experiments. The fibroblast-injection model does not simulate the time course of PVR development in humans. Furthermore, the optimal formulation, delivery method, and dosing regimen of the codrug was not defined. This study was designed to evaluate the efficacy and pharmacokinetics of an improved formulation of a codrug consisting of triamcinolone acetonide and 5-fluorouracil (TA/5-FU) in the treatment of a refined rabbit model of PVR.MATERIALS AND METHODSAll animal experiments were conducted in accordance with the guidelines set forth by the Association for Research in Vision and Ophthalmology, Rockville Pike, Md, for the use of animals in ophthalmic and vision research. Experimental manipulations were performed on right eyes only. New Zealand white rabbits of either sex weighing approximately 1.5 to 2 kg were used for this study.Experiments were divided into 3 parts as follows. Part 1 (41 rabbits): the therapeutic efficacy of 2.5 mg (group 1) or 10 mg (group 2) of TA/5-FU codrug powder injected intravitreally was compared with controls in the treatment of experimental PVR.Part 2 (30 rabbits): the therapeutic efficacy of one 2.5-mg TA/5-FU codrug pellet (group 3) or three 3.3-mg pellets (group 4) implanted intravitreally was compared with controls in the treatment of experimental PVR.Part 3 (15 rabbits): in vivo pharmacokinetics and toxic effects of the 10-mg TA/5-FU codrug suspension in normal rabbit eyes were determined. The in vitro release rates of the codrug powder and pellet were also measured.PREPARATION OF TA/5-FU POWDER FOR INJECTIONA preweighed quantity of codrug powder was placed in a Teflon injection catheter, then connected to a syringe that contained 0.85 mL of hyaluronic acid (Provisc, Alcon, Fort Worth, Tex). The hyaluronic acid was used to mechanically push codrug powder through the injection catheter into the vitreous cavity through a sclerotomy.PREPARATION OF TA/5-FU PELLETS FOR IMPLANTATIONImplantable sustained-release pellets containing 2.5 mg or 3.3 mg of the TA/5-FU codrug were prepared by direct compression of the powdered codrug into a 1.5-mm disk with a customized press (Parr Instruments, Moline, Ill).PVR INDUCTIONSeventy-one New Zealand white rabbits underwent pars plana lensectomy, core vitrectomy, and penetrating retinal endodiathermy to induce PVR as previously described.Briefly, animals were anesthetized with an intramuscular injection of 0.3 mL of ketamine hydrochloride (100 mg/mL; Fort Dodge Laboratories, Fort Dodge, Iowa) and 0.1 mL of xylazine hydrochloride (100 mg/mL; Miles Inc, Shawnee Mission, Kan) per kilogram of body weight. A 5-mm peritomy was made at the superotemporal and superonasal quadrant of the right eye. Sclerotomies were created with a 19-gauge microvit-reoretinal blade 1 to 2 mm posterior to the limbus in the superonasal and superotemporal quadrants. An infusion light pipe was inserted through the first sclerotomy and an ultrasonic fragmatome was inserted through the second sclerotomy. Lensectomy was performed with the ultrasonic fragmatome, and the remnants of lens capsule were removed with a side-cutting vitreous cutter. The vitreous cutter and infusion light pipe were used to perform a 2-port core vitrectomy. Two endodiathermy spots were applied in 3 equatorial retinal quadrants (inferior to optic disc, inferotemporal quadrant, and inferonasal quadrant) with an endodiathermy unit (model TR-3000, MIRA, Waltham, Mass) in continuous mode with the power setting at 3 to 4. A full-thickness retinal hole with a surrounding white rim was created after application of the endodiathermy probe to the retina for approximately 1 second. A sclerotomy was enlarged to a length of 2.5 mm with a keratome. Rabbits in treatment groups underwent codrug powder injection or pellet implantation through the enlarged sclerotomy. Control rabbits received hyaluronic acid injection or sham operation alone.DRUG INJECTION OR IMPLANTATIONIn part 1, the codrug powder, prepared as described earlier, was injected into the midvitreous cavity of the right eye after endodiathermy under direct view with an operating microscope. To avoid codrug reflux after injection caused by increased intraocular pressure, the infusion light pipe was removed from the sclerotomy to allow spontaneous egress of fluid during the injection. Control rabbits received an injection of hyaluronic acid alone. In group 1, the 2.5-mg TA/5-FU codrug powder was compared with controls. Twenty-two rabbits were used for this portion of the study, including 10 treated and 12 control rabbits. In group 2, 10-mg TA/5-FU codrug powder was compared with controls. Nineteen rabbits were used for this portion of the study, including 9 treated rabbits and 10 control rabbits.In part 2, the codrug pellet, prepared as described earlier, was implanted into the vitreous base of the eyes of the experimental rabbits after endodiathermy. The implanted pellet remained in the vitreous base, and did not migrate posteriorly. Control rabbits underwent a sham operation alone. In group 3, implantation of one 2.5-mg pellet of TA/5-FU codrug was compared with controls. In group 4, implantation of three 3.3-mg pellets of TA/5-FU codrug was compared with controls. Thirty rabbits were used for this portion of the study, including 11 that received 1 pellet, 10 that received 3 pellets, and 9 controls. The sclerotomies were closed with 7-0 polyglactin 910 sutures. One drop (&ap;20 µL) of 0.3% gentamicin solution was instilled into the eye after surgery for infection prophylaxis. To maintain pupil dilation, 1 drop of 1% atropine solution was instilled daily for 1 week, then every other day for a second week.PVR GRADING AND CLINICAL EXAMINATIONPupils were dilated with 1 drop each of 2.5% phenylephrine and 1% tropicamide. The PVR grade was scored in a masked fashion weekly for 12 weeks by 2 independent observers using a classification system previously described by Hida and associates(Table 1) The PVR grade for each hemiretina was determined, and the 2 grades were then summed to obtain a total eye score (Figure 1). On week 12, all animals were killed under anesthesia using an intracardiac injection of pentobarbital sodium (Anpro Pharmaceuticals, Arcadia, Calif).Clinical Grading of Proliferative VitreoretinopathySee table graphicFigure 1.Fundus photograph showing examples of different grades of proliferative vitreoretinopathy. A, Codrug-treated eye with attached retina and normal medullary ray and optic nerve (grade 0). B, Control eye showing dragging of medullary ray and severe tractional elevation of the retina (>2 disc diameters) (grade 4). C, Control eye showing bullous detachment of the retina (grade 5). The retina is drawn over the optic disc and there is a large retinal break inferiorly.IN VIVO PHARMACOKINETICSThe right eye only of 12 New Zealand white rabbits (weight, 1.5-2.0 kg) was used for pharmacokinetic analysis. Anesthesia was performed with an intramuscular injection of ketamine and xylazine as described earlier. A 1.5-mm sclerotomy was created 1.0 mm posterior to the limbus in the superior temporal quadrant. To soften the eye, a 26-gauge needle was inserted into the anterior chamber at the limbus and 0.1 mL of aqueous removed. Ten milligrams of the TA/5-FU codrug was suspended in 0.1 mL of hyaluronic acid (Healon, Pharmacia Inc, Columbus, Ohio) and injected into the midvitreous through a 19-gauge needle inserted through the sclerotomy. The tip of the needle was directly viewed with an operating microscope during injection of the codrug. The sclerotomy was closed with 7-0 polyglactin 910 sutures. After injection, the codrug was initially suspended in the midvitreous. Although it remained in this location in a few eyes, the codrug settled into the inferior vitreous cavity in most eyes.Animals were killed at 1, 7, 42, and 84 days. Three animals were killed at each time point by intracardiac injection of pentobarbital. The eyes were enucleated and immediately frozen at −80°C. The tissues were then prepared for assay as described below.DRUG LEVEL ANALYSISThe vitreous was dissected from the frozen globe. Triamcinolone, 5-fluorouracil, and intact codrug levels in 100 µL of vitreous were determined. Assays were performed using a fully automated high-pressure liquid chromatography system (Hitachi Scientific Instruments, Mountain View, Calif) with a C-18 reverse-phase column with a C-18 guard column. Acetonitrile and 0.02% sodium acetate (pH 4.0) were used in the mobile phases. Detection was by UV light at 266 nm for 5-fluorouracil and 234 nm for triamcinolone and intact codrug.ASSESSMENT OF TOXIC EFFECTSToxic effects of the TA/5-FU codrug were determined on eyes used for the pharmacokinetic analysis (described above). Slitlamp examination and indirect ophthalmoscopy were performed immediately prior to death, ie, at 1, 7, 42, and 84 days.Scotopic electroretinography (ERG) was performed in both eyes of 3 additional rabbits prior to injection of 10 mg of codrug powder in the right eye. Postinjection ERG was performed at 14, 28, and 63 days. Electroretinography examinations were recorded with the use of contact lens electrodes (ERG-Jet, Universo SA, La Chaux-de-Fonds, Switzerland) with a 2-channel clinical signal averager (No. 5200, Cadwell, Kennewick, Wash) and a Ganzfeld flash unit (VPA-10, Cadwell). Scotopic ERGs, obtained after at least 30 minutes of dark adaptation, were elicited at 0.34 Hz. For each ERG, 20 stimulus presentations were averaged. To minimize the effect of individual and daily variation on the ERG, the ratio of the B-wave amplitude of the experimental (right) eye to the B-wave amplitude of the control (left) eye was determined. When the amplitude of the experimental and control eyes are equal, the ratio equals 1. A decrease in the ratio reflects a relative decrease in the B-wave amplitude of the experimental eye.The rabbits used for ERG analysis were killed at 65 days for histopathologic analysis. The codrug-injected eyes were immediately enucleated and a 3–clock-hour circumferential incision was created 1.0 mm posterior to the limbus. The globes were fixed by immersion in 2% glutaraldehyde in 0.1-mol/L sodium cacodylate buffer (pH, 7.4) and processed in paraffin. Sections were stained with hematoxylin-eosin and examined by light microscopy.IN VITRO PHARMACOKINETIC STUDYDrug release from the 2 formulations of codrug (2, 5, and 10 mg of codrug powder; 1, 4, and 8 mg of codrug pellet) were determined by placing the codrug into a microcentrifuge tube containing 1.0 mL of 0.1-mol/L phosphate buffer solution (pH, 7.4; 37°C). Every 24 hours the tubes were centrifuged and 0.5 mL of the supernatant was removed for analysis by reverse-phase high-pressure liquid chromatography as described earlier. Then, 0.5 mL of fresh buffer was added and the determination continued for 32 days.STATISTICAL ANALYSISA Mann-Whitney Unonparametric test was used to compare the difference in median clinical grade of PVR between experimental and control groups. The χ2test was used to compare the difference in the number of eyes with retinal detachment between experimental and control animals. A paired 2-tailed ttest was used to compare the ERG B-wave amplitude ratio before and after codrug injection.RESULTSCLINICAL OBSERVATIONSIn part 1 (TA/5-FU codrug powder), the severity of PVR was significantly less in both experimental groups as compared with controls from week 4 to week 12 (P<.01 in group 1, P<.05 in group 2, Mann-Whitney Utest) (Figure 2, A and B). In group 1 (2.5 mg of codrug powder), the percentage of eyes with moderate tractional detachment (grade 3 in at least 1 ray) or worse was greater in the control group (50%) than in the treated group (0%) at week 8 (P<.05, χ2test) (Figure 3, A). In group 2 (10 mg of codrug powder), the percentage of eyes with moderate tractional detachment or worse was greater in the control group than in the treated group at all time points, but the difference was not statistically significant (Figure 3, B).Figure 2.Median clinical grade of proliferative vitreoretinopathy as a function of time. A, Group 1 (2.5 mg of triamcinolone acetonide and 5-fluorouracil [TA/5-FU] codrug powder). B, Group 2 (10 mg of TA/5-FU codrug powder). C, Group 3 (one 2.5-mg TA/5-FU pellet). D, Group 4 (three 3.3-mg TA/5-FU pellets). Median grade was 0 at time points that do not show a data bar.Figure 3.The percentage of eyes with moderate tractional detachment or worse as a function of time. A, Group 1 (2.5 mg of triamcinolone acetonide and 5-fluorouracil [TA/5-FU] codrug powder). B, Group 2 (10 mg of TA/5-FU codrug powder). C, Group 3 (one 2.5-mg TA/5-FU pellet). D, Group 4 (three 3.3-mg TA/5-FU pellets). The percentage of eyes with moderate tractional detachment or worse was 0 at time points that do not show a data bar.In part 2 (TA/5-FU codrug pellet), both the severity of PVR grade and percentage of eyes with moderate tractional detachment or worse were significantly less in group 4 (three 3.3-mg pellets) than in controls from week 5 to week 12 (P<.01) (Figure 2, D and Figure 3, D). In group 3 (one 2.5-mg pellet), the severity of PVR was less in treated eyes than in controls at all time points, but the difference was not statistically significant (P=.07 at week 11 and 12, Mann-Whitney Utest) (Figure 2, C). Nevertheless, the percentage of eyes with moderate tractional detachment or worse was statistically significantly less in treated eyes (40%) than in controls (90%) at week 8 and thereafter (P=.05, χ2test) (Figure 3, C).IN VIVO PHARMACOKINETICSIn eyes receiving the 10-mg codrug suspension, the vitreous concentration of free TA remained above 200 µg/mL for the first 6 weeks of the study, and then declined to 36 µg/mL by the 12th week. 5-Fluorouracil levels were above 50 µg for the first 6 weeks and remained above 10 mg/mL thereafter (Figure 4). The amount of intact codrug in the vitreous cavity gradually declined from 10 µg to 2.9 mg by week 6 and was still measurable (0.47 mg) by week 12. Ophthalmoscopy was used to determine whether codrug powder was visible clinically. At week 12, a moderate amount of codrug powder was visible in the vitreous cavity of 2 eyes and a trace amount was visible in 1 eye.Figure 4.Concentration of triamcinolone acetonide and 5-fluorouracil in rabbit vitreous as a function of time after injection of 10 mg of triamcinolone–5-fluorouracil codrug suspension. Data represent mean±SD; n=3 at each time point.IN VITRO PHARMACOKINETICSIn vitro codrug powder (2, 5, and 10 mg) provided sustained release of 5-fluorouracil for up to 33 days (Figure 5, A). The surface areas of the 1-, 4-, and 8-mg codrug pellet are 9.0, 21.8, and 43.5 mm2, respectively. The 5-fluorouracil release from codrug pellets (1-, 4-, and 8-mg) was proportional to their surface area and constant over this period (Figure 5, B and C).Figure 5.A, In vitro release of 5-fluorouracil from triamcinolone acetonide and 5-fluorouracil (TA/5-FU) codrug powder placed in 0.1-mol/L phosphate buffer. Data represent mean±SD; n=3 at each time point. B, In vitro release of 5-fluorouracil from TA/5-FU codrug pellets placed in 0.1-mol/L phosphate buffer. C, In vitro release rate of codrug pellets (1, 4, and 8 mg) plotted as a function of pellet surface area. Data represent mean±SD; n=3 at each time point.CODRUG TOXICITYThere was no evidence of toxic effects based on the clinical examinations. Specifically, no inflammation, cataract formation, or retinal abnormalities developed.Electroretinograms showed no evidence of codrug toxic effects. The B-wave amplitudes remained normal throughout the study. Ratios of the ERG B-wave amplitudes of the injected eye to the B wave of the contralateral (control) eye were not statistically different when preinjection values were compared with postinjection values (P=.27 at day 63, paired ttest) (Figure 6).Figure 6.Representative scotopic electroretinography in eye receiving 10 mg of triamcinolone acetonide and 5-fluorouracil codrug. The ratios of the electroretinographic B-wave amplitude of the right eye (injected) to the left eye (control) were not statistically different when preinjection values (left) were compared with postinjection values (right) at day 63 (P=.27).On histopathologic analysis, the ciliary body and retina were normal by light microscopic examination (Figure 7). No sign of retinal necrosis, photoreceptor cell loss, cystic degeneration, inflammatory cell infiltration, or hypocellularity of nuclear layers was observed.Figure 7.Histopathologic appearance of the retina at medullary ray (left) and ciliary body (right) at week 9 after injection of 10 mg of triamcinolone acetonide and 5-fluorouracil codrug (hematoxylin-eosin, ×250).COMMENTThe results of our study demonstrate that injectable or implantable TA/5-FU codrug effectively inhibits the progression of PVR in a rabbit model. Both the severity of PVR grade and percentage of eyes with moderate tractional detachment or worse were significantly less in experimental eyes than control eyes in all groups. There was no evidence of drug-related toxic effects by ERG or on clinical or histopathologic examination of eyes containing the TA/5-FU codrug.Ideally, sustained drug-delivery systems should release the drug over a period that corresponds to the duration of disease activity. The earliest signs of PVR, cells in the vitreous cavity and subretinal space, are visible in most patients soon after rhegmatogenous retinal detachmentProgression to formation of fibrocellular membranes and subsequent cell-mediated contraction of these membranes occurs over the ensuing 6 to 12 weeks after surgery to repair the retinal detachment.The codrug systems described in the current report produced a therapeutic effect for the duration of the 12-week observation period. This effect was paralleled by sustained intravitreal levels of triamcinolone and 5-fluorouracil. During the first 6 weeks the vitreous concentration of free triamcinolone remained above the concentration of drug required to produce 50% inhibition of fibroblast cell growth (the 50% inhibition level for triamcinolone is 150 µg/mL).The concentration of free 5-fluorouracil remained above the 50% inhibition level for fibroblast cell growth (0.3 µg/mL) for the entire 12-week duration of the experiment.Similarly, the in vitro pharmacokinetic analysis showed that the TA/5-FU codrug suspension produced sustained drug release over an extended period. Together, these data suggest that the corticosteroid–5-fluorouracil codrug pharmacokinetic profile would be advantageous in the treatment of PVR, a disease that typically runs its course over a 6- to 12-week period.The injectable codrug powder had a different pharmacokinetic profile than the compressed codrug pellet. As demonstrated by the in vitro studies, drug release rate is directly proportional to the surface area. It is possible to achieve a much higher surface area with the codrug powder than with the compressed pellet. Thus, for an equivalent quantity of codrug over a given period, the amount released from the codrug powder is higher than that released from the pellet. Furthermore, the codrug powder followed logarithmic rather than linear release kinetics; this produced high initial drug levels in the vitreous, which subsequently logarithmically declined. In contrast, the codrug pellet released the drug in a linear manner. We hypothesize that these pharmacokinetic profiles accounted for the observation that the 2.5 mg of codrug powder more effectively inhibited PVR in the rabbit eye than the 2.5-mg codrug pellet. It is likely that the initial pulse of triamcinolone and 5-fluorouracil provided by the powdered codrug rapidly suppressed cellular proliferation and inflammation, and that lower drug levels were necessary to maintain suppression until cellular proliferation ceased. In contrast, the constant drug levels obtained with the 2.5-mg codrug pellet may have been insufficient to completely suppress cellular proliferation and inflammation at a time when disease activity was maximal.The powdered codrug has several potential advantages over the compressed pellet and polymeric delivery devices. As discussed earlier, the kinetic release profile of the powdered codrug may be advantageous in the treatment of PVR. In addition, because the powdered codrug is given as an injection, it is possible to administer the drug without creating a large surgical wound. In contrast, the codrug pellet described in the current report and polymeric devices described previouslymust be inserted through a relatively large surgical wound. Despite these advantages, if the powdered codrug were used to treat PVR, drug dispersion into the vitreous cavity could temporarily diminish the physician's view of the patient's retina during postsurgical follow-up examinations. In contrast, the codrug pellet provides local drug delivery without obscuring the retinal view. Both formulations are improvements over the polymeric device described in our previous report; the compressed pellet and codrug powder serve as their own delivery devices and no residual foreign body remains in the eye after the codrug has disappeared. In contrast, the nonerodible device described in our previous reports remains within the eye permanently.The technique used to deliver the injectable form of the codrug is an improvement over that described in our previous report.In the previous experiments, the codrug powder was suspended in balanced isotonic saline solution or hyaluronic acid prior to injection. After injection of this suspension, there was frequently residual codrug adherent to the walls of the syringe. Thus, the quantity of injected codrug was somewhat variable. In the current study, hyaluronic acid was used to mechanically push the desired quantity of codrug powder out of the syringe into the vitreous cavity. With this technique, there was no residual codrug in the syringe. Thus the quantity of the codrug injected was reproducible and accurate.In previous investigations, we chose the fibroblast injection model of PVR to test the efficacy of a corticosteroid–5-fluorouracil conjugate.However, this model has several limitations. For example, PVR develops more rapidly in the fibroblast-injection model than in humans.Unlike human PVR, in the fibroblast-injection model PVR is not initiated by a retinal tear and neovascularization is a prominent component. Furthermore, drug therapy may inhibit injected fibroblasts, which would otherwise induce proliferation of endogenous cells. Finally, because of the severe nature of PVR in this model, lack of drug efficacy does not necessarily mean that the drug would be ineffective in humans. For these reasons, in this investigation we chose a refined model of PVR that has several advantages over that used previously. In the refined model, first described by Iverson and associates,PVR is produced by creating full-thickness retinal defects accompanied by breakdown of the blood-retinal barrier induced by lensectomy, vitrectomy, and penetrating diathermy. In this model, proliferation is induced only by endogenous cells, neovascularization is not a prominent component, and PVR occurs reproducibly in more than 90% of animals. Most importantly, unlike the rapid time course of PVR observed in the fibroblast-injection model, with the refined model the time course for development of PVR (4-12 weeks) is very similar to that observed in humans. This time course allowed us to test codrug efficacy over a protracted period, which was not possible with the fibroblast-injection model.The TA/5-FU codrug conjugate was not toxic to the normal rabbit eye. We chose to evaluate the safety of the codrug in normal animal eyes rather than eyes with retinal detachment and PVR. It is recognized that the potential toxic effects may differ in normal eyes and in eyes with a retinal detachment and PVR. However, eyes with retinal detachment often develop decreased intraocular pressure, loss of ERG B wave, retinal edema, and loss of photoreceptor outer segments—changes that can mimic the toxic effects from an intraocular drug. Therefore, because it may be virtually impossible to distinguish drug effects from those induced by the retinal detachment, experiments conducted in normal eyes have generally been considered an appropriate means to evaluate toxic effects.Clinically, one could envision several possible means of delivering the TA/5-FU codrug for prophylaxis or treatment of PVR. Evaluation of toxic effects in the primate eye would be necessary before beginning human clinical trials. Assuming a lack of toxic effects, one could consider injecting the codrug prophylactically into the vitreous cavity at the time of primary scleral buckle to repair a rhegmatogenous retinal detachment. Alternatively, the codrug might be placed in the eye at the conclusion of vitrectomy to repair a primary or recurrent rhegmatogenous retinal detachment in the presence or absence of PVR. In this situation, gas is often used as a vitreous substitute to provide an extended retinal tamponade. The pharmacokinetics of the codrug in an eye containing gas are unknown. Although they are beyond the scope of the current report, experiments to determine the toxic effects of the codrug in primates and the pharmacokinetics of the codrug in a gas-filled eye are under way in our laboratory.RMachemerProliferative vitreoretinopathy (PVR): a personal account of its pathogenesis and treatment.Invest Ophthalmol Vis Sci.1988;29:1771-1783.The Silicone Study GroupProliferative vitreoretinopathy.Am J Ophthalmol.1985;99:593-595.WFRachelTCBurtonChanging concepts of failures after retinal detachment surgery.Arch Ophthalmol.1979;97:480-483.YTanoDChandlerRMachemerTreatment of intraocular proliferations with intravitreal injection of triamcinolone acetonide.Am J Ophthalmol.1980;90:810-816.DBChandlerGRozakisEDejuanRMachemerThe effect of triamcinolone acetonide on a refined experimental model of proliferative vitreoretinopathy.Am J Ophthalmol.1985;99:686-690.MSunalpPWiedemannNSorgenteSJRyanEffects of cytotoxic drugs on proliferative vitreoretinopathy in the rabbit cell injection model.Curr Eye Res.1984;3:619-623.JAKhawlyPSaloupisDLHatchellRMachemerDaunorubicin treatment in a refined experimental model of proliferative vitreoretinopathy.Graefes Arch Clin Exp Ophthalmol.1991;229:464-467.MLemorJHYeoBMGlaserOral coldhicine for the treatment of experimental traction retinal detachment.Arch Ophthalmol.1986;104:1226-1229.PWiedemannMKirmaniMSantanaControl of experimental massive periretinal proliferation by daunomycin: dose-response relation.Graefes Arch Clin Exp Ophthalmol.1983;220:233-235.PWiedemannNSorgenteCBekhorDaunomycin in the treatment of experimental proliferative vitreoretinopathy.Invest Ophthalmol Vis Sci.1985;26:719-725.MSBlumenkranzAOphirAJClaflinAHajekFluorouracil for the treatment of massive periretinal proliferation.Am J Ophthalmol.1982;94:458-467.ASBergerCKChengPAPearsonIntravitreal sustained release corticosteroid-5-fluorouracil conjugate in the treatment of experimental proliferative vitreoretinopathy.Invest Ophthalmol Vis Sci.1996;37:2318-2325.DIversonWDaileyMHartzerPars plana lensectomy, vitrectomy and transvitreous diathermy in the rabbit eye: a model of PVR.Invest Ophthalmol Vis Sci.1991;32(suppl):856.THidaDBChandlerSMShetaClassification of the stages of proliferative vitreoretinopathy in a refined experimental model in the rabbit eye.Graefes Arch Clin Exp Ophthalmol.1987;225:303-307.LFeeneyRBurnsRMixonHuman subretinal fluid.Arch Ophthalmol.1975;93:62-69.BMGlaserMLemorPathobiology of proliferative vitreoretinopathy.In: Ryan SJ, ed. Retina.St Louis, Mo: Mosby–Year Book; 1988:369-383.HGrossfieldCRaganAction of hydrocortisone on cells in tissue culture.Proc Soc Exp Biol Med.1954;86:63-68.ARuhmannDBerlinerEffect of steroids on growth of mouse fibroblasts in vitro.Endocrinology.1965;76:916-927.MSBlumenkranzAClaflinASHajekA pharmacologic approach to non-neoplastic intraocular proliferation.Invest Ophthalmol Vis Sci.1981;20(suppl):200.MSBlumenkranzAClaflinASHajekSelection of therapeutic agents for intraocular proliferative disease: cell culture evaluation.Arch Ophthalmol.1984;102:598-604.DFMartinDJParksSDMellowTreatment of cytomegalovirus retinitis with an intraocular sustained-release ganciclovir implant: a randomized clinical trial.Arch Ophthalmol.1994;112:1531-1539.GJJaffeASBergerDPHainesworthPAshtonIntravitreal sustained release triamcinolone/5-FU conjugate suspension in the treatment of experimental PVR.Invest Ophthalmol Vis Sci.1995;36:S161.ASBergerCKChengPAPearsonIntravitreal sustained release dexamethasone/5-FU device in the treatment of experimental PVR.Invest Ophthalmol Vis Sci.1994;35(suppl):1923.PAlgevereEKockExperimental fibroplasia in rabbit vitreous.Albrecht Von Graefes Arch Klin Exp Ophthalmol.1976;199:215-222.DBChandlerFHQuansahRMachemerA refined experimental model for proliferative vitreoretinopathy.Graefes Arch Clin Exp Ophthalmol.1986;224:86-91.GJJaffePAPearsonProliferative vitreoretinopathy: biology and pharmacology.In: Weinberg DV, Jampol LM, eds. Ophthalmic Pharmacology.New York, NY: Raven Press; 1997:417-428.Accepted for publication August 19, 1997.This study was supported by the Adler Foundation (Dr Jaffe), National Institutes of Health (NIH), Bethesda, Md, grant EYO-9106 (Dr Jaffe), NIH grant EYO-11041 (Dr Ashton), NIH core grant P30 EYO5722 Controlled Delivery Systems (Drs Ashton and Jaffe), The Heed Foundation (Drs Khawly and Hainsworth), and an unrestricted award from Research to Prevent Blindness Inc, New York, NY. Dr Jaffe is a Research to Prevent Blindness Lew R. Wasserman Merit Award recipient. Dr Ashton has applied for a patent on the codrug technology.Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Fla, April 23, 1996.Reprints: Glenn J. Jaffe, MD, Box 3802, Duke University Eye Center, Durham, NC 27710.

1998 Archives of Ophthalmology

doi: 10.1001/archopht.116.1.77

Due to errors in cropping of figures during processing for publication in the article titled "A Comparison of Retinal Morphology Viewed by Optical Coherence Tomography and by Light Microscopy," in the November issue of the ARCHIVES (Arch Ophthalmol. 1997;115:1425-1428), the upper portion of the top image in Figure 1 is missing and the left and right parts of Figure 2 are incorrectly matched. Figure 1 and Figure 2 are reprinted correctly here. The Journal regrets the errors. Figure 1. View LargeDownload Optical coherence tomography (OCT) of a section of retina (top) and its corresponding histologic section stained with methylene blue (bottom). Note the laser lesion (created with an estimated retinal spot size of 200 µm) visible in both images (between solid arrowheads). The laser lesion images were aligned for the comparison of adjacent retinal layers as seen in Figure 2. The 250-µm scale bar holds for both the x- and y-coordinates of the OCT image. The open arrows indicate the broad layer of relative low reflectivity corresponding to the photoreceptor nuclei and inner and outer segments. Figure 2. View LargeDownload Optical coherence tomography (OCT) of the macula (left) and its corresponding histologic section stained with methylene blue (right). An outline of the retinal layers from the corresponding light micrograph is superimposed over the OCT image. Note the coalescence of layers entering the fovea. The open arrows indicate the broad layer of relative low reflectivity that corresponds to the nuclear layer and the inner and outer segments of the photoreceptors. This layer widens in the fovea.