Advances Help Researchers To See The Cornea More Clearly

The cornea used to be the Rodney Dangerfield of the eye. Once jokingly dismissed as a mere dust cover by scientists who study the retina, the cornea is gaining new respect as research reveals its complexities. IMPETUS FOR INTEREST: UC-Davis's Mark Mannis maintains that advances in technology and molecular and cell biology are spurring interest in cornea research. "Probably the reason for the interest, first of all, is the burgeoning technology in refractive surgery and in implantation devices," explains Mark J. Mannis, a professor of ophthalmology at the University of California, Davis, Medical Center in Sacramento, and editor-in-chief of the journal Cornea. "So on one side there's a technological impetus to increase our interest in [the] cornea. And the other is probably an enhanced understanding of molecular biology and cell biology, which is now being applied to [the] cornea, both diagnostically and therapeutically. . . . I think that the new initiatives in modulating the way the cornea heals, both from the point of view of refractive surgery and of the replacement of cells in eyes that have been severely damaged from burns and other types of disorders, are extremely exciting." The National Eye Institute (NEI) apparently agrees. NEI funding for cornea research increased from $33.9 million in fiscal year 1995 to $36.6 million in FY96 and to $39 million in FY97, up 15 percent in two years. And cornea research is making up a larger percentage of the total NEI budget, increasing from 11.3 percent of the institute's $300 million budget in FY95 to 11.8 percent of the institute's $331 million budget in FY97. The increased interest and increased funding appear to be translating directly into an enhanced understanding of this deceptively simple tissue. "The cornea seems to be marvelously adapted," remarks Jerry Y. Niederkorn, who holds the George A. and Nancy P. Shutt Professorship in Medical Science at the University of Texas (UT) Southwestern Medical Center at Dallas. "If you think about it, it's a very simple tissue. You have just three major components-the epithelium, the stroma, and the endothelium-and you don't have any other structures involved. You don't have blood vessels. You don't have hair follicles. You don't have fat glands. And so it's an elegantly simple tissue, but it performs an extraordinarily complicated function in being perfectly clear and providing a perfectly refractive medium for the transmission of light." Harold F. Spalter, a professor of clinical ophthalmology at Columbia University College of Physicians and Surgeons, recently moderated a "Science Writers in Ophthalmology" seminar in which several cornea scientists discussed the latest advances in cornea research. One topic that came up during the seminar, sponsored by Research to Prevent Blindness (RPB), a New York-based foundation, was the divisions between "retinologists" and "cornea people." "The eye provides its own landmarks of separation and friendly borders that we don't tread over very much, like borders between countries," Spalter noted. "Not that we necessarily need passports to examine other portions of the geography, but there is a friendly division between so-called anterior segment ophthalmologists-dealing with the cornea, the superficial layers of the eye, and the lens-and those of us who then attack the problems that start behind the lens of the eye in the vitreous cavity and the retina." Although some scientists seem to regard retina studies as somehow more glamorous than cornea research, Niederkorn points out that "no matter how complicated and sophisticated the neuronal components of the retina are, they're useless without a clear cornea." Unfortunately, several conditions can compromise that clarity. One of the most important is ulcerative/infectious keratitis (UK), which is often a consequence of contact lens wear. It's a bacterial infection that H. Dwight Cavanagh, the W. Maxwell Thomas Chair Professor at UT Southwestern Medical Center, says can come on like "lightning striking out of the blue" and can lead to blindness. The annual incidence of UK is 4 in 10,000 for people who wear their lenses only during the day and 20 in 10,000 for people who sleep in their lenses. Sixty million people worldwide wear contact lenses. Although these are fairly high rates of adverse reactions, "we sat for a decade stagnant, still in the water, without major progress in solving and resolving this vexatious problem," Cavanagh points out. "The problem was that we had no outcome measure in research that would predict the keratitis occurrence later in large numbers of patients." Animal experimentation provided some clues, revealing, for example, that contact lenses make it easier for bacteria such as Pseudomonas aeruginosa to bind to corneal epithelial cells, and this binding is the first stage in infection. But it's clearly unethical to place infectious bacteria into human eyes. Cavanagh managed to find a way around this problem. He and his coworkers developed an eye-irrigation device that gently and painlessly washes epithelial cells from the surface of the human cornea. These cells-obtained from people who have been wearing a variety of contact lenses-can then be tested for bacterial binding in culture. Cavanagh determined that the less oxygen permeable the lens, the more bacteria bound to the cells (M. Imayasu et al., Ophthalmology, 101:371-88, 1994). Moreover, in a study that has not yet been published, Cavanagh tested new "superlenses" that are being developed by three manufacturers. The compositions of those lenses are "closely held trade secrets," he notes, but they have a very high water content with "pinches of silicon fluoroacrylate," which greatly increases oxygen permeability. Bacterial binding to washed cells did not increase even after people had been wearing these superlenses continuously for three months. "So it looks like if you get a high enough oxygen-transmissible lens, it fits like a regular soft lens, it looks like a soft lens, hopefully it's going to cost the same as a regular soft lens, . . . it gives the good vision of a soft lens, it gives the comfort the public wants, but isn't going to alter the biology of the corneal surface over time with lens wear," concludes Cavanagh. He adds that a Food and Drug Administration-approved clinical trial of at least one of these extended-wear lenses is about to begin. Research on extended-wear contact lenses has the goal of making temporary vision correction more permanent. Photorefractive surgery takes a more direct approach to making permanent vision corrections, but surgery to correct myopia is still imperfect. Radial keratotomy (RK), a once- popular technique in which tiny incisions arranged like the spokes of a wheel were made in the cornea, has largely been supplanted by two laser techniques. In photorefractive keratectomy (PRK), first the surgeon scrapes the cornea's epithelial layer, and then an eximer laser sculpts the underlying stroma. In laser in situ keratomileusis (LASIK), first the surgeon slices off the outer layer of the cornea, then the laser sculpts the stroma, and finally the surgeon replaces the outer layer. FOOLING KETRATOCYTES: Steven Wilson is trying inhibit apoptosis to eliminate wound-healing responses that can change the shape of the cornea. Although the laser sculpting in both PRK and LASIK is controlled by computer to provide the precise changes in corneal shape calculated to produce perfect vision, a significant minority of patients do not achieve this goal. According to Steven E. Wilson, medical director of the Refractive Eye Laser Center at the Cleveland Clinic Foundation, only 80 percent of patients have 20/20 vision one year after PRK. Between 95 percent and 98 percent have their vision corrected to 20/40 or better. Wilson thinks there's no fundamental problem with the surgical techniques. Instead, "as these lasers become much more precise, we are now at the point where the real difference between patients is no longer the laser. It's the wound-healing differences between individual patients. And if we are able to develop ways to control this wound-healing difference, we can make these procedures even more predictable than they are now." Wilson's research demonstrates that these variations in wound healing depend on complex interactions between epithelial cells on the outer surface of the cornea and underlying fibroblast cells called keratocytes. Epithelial cells damaged by the surgeon's knife release cytokines that trigger apoptosis-programmed cell death-in the keratocytes. The dead keratocytes are replaced within two or three days, but the new keratocytes are "activated," and they synthesize extra collagen, hyaluronic acid, and growth factors, all of which can lead to a change in the shape of the cornea (S.E. Wilson et al., Investigative Ophthalmology and Visual Sciences, 37:1582-92, 1996). "If we can inhibit this initial apoptosis in response to the cytokines from the epithelium, we can eliminate a lot of the subsequent wound-healing response," notes Wilson hopefully. "In other words, what we want to do is to fool the underlying keratocytes into never knowing that the tissue above them-the epithelium and anterior stroma-was ever damaged. "Why would a system like this exist in the cornea?" Wilson asks. "We hypothesized early on that this was an antiviral system." He suggests that when a virus infects the cornea, the death of anterior keratocyte cells leaves nowhere for it to spread. When corneal damage due to disease or injury becomes severe, the only alternative is transplantation. Corneal transplants are now routine. Niederkorn notes that such surgery is "the oldest and the most common type of solid-tissue transplantation performed. In the United States alone there are greater than 42,000 corneal transplants performed each year." According to Niederkorn, only 10 percent to 15 percent of these transplants undergo immunological rejection. But in a mouse model of corneal transplants, Niederkorn has developed a novel approach he hopes will reduce the number of rejected corneas even further. American Academy of Ophthalmology (AAO) 655 Beach St. San Francisco, Calif. 94109-1336 (415) 561-8500 ¥ Fax: (415) 561-8533 http://www.eyenet.org/ 22,000 members President: Stephen A. Obstbaum Journal: Ophthalmology Don Minckler, Editor-in-Chief Doheny Eye Institute 1450 San Pablo St. Los Angeles, Calif. 90033-4666 (213) 342-6350 - Fax: (213) 342-6359 http://www.eyenet.org/ophthalmology Association for Research in Vision and Ophthalmology (ARVO) 9650 Rockville Pike Bethesda, Md. 20814-3998 (301) 571-1844 ¥ Fax: (301) 571-8311 http://www.faseb.org/arvo/ 10,200 members President: Paul L. Kaufman Journal: Investigative Ophthalmology and Visual Science Gerald Chader, Editor-in-Chief 9650 Rockville Pike, Suite 1514 Bethesda, Md. 20814-3998 (301) 571-1860 or 5739 Fax: (301) 571-8315 http://www.faseb.org/arvo/iovs.htm Castroviejo Cornea Society c/o Edward J. Holland, Executive Secretary-Treasurer Box 79 UMHC 420 Delaware St., S.E. Minneapolis, Minn. 55455-0501 (612) 625-3233 or (800) 937-4393 430 members President: Jay H. Krachmer Journal: Cornea Mark J. Mannis, Editor-in-Chief University of California Davis Medical Center 1611 Alhambra Blvd. Sacramento, Calif. 95816 (916) 734-6086 http://www.lippincott.com/cornea/ Research to Prevent Blindness (RPB) 645 Madison Ave. New York, N.Y. 10022-1010 (800) 621-0026 or (212) 752-4333 Fax: (212) 688-6231 RPB provided more than $8 million in support of vision science in 1996. Inbred mice are a good model system for studying corneal transplants, since they reject 100 percent of corneal transplants from unrelated donors when anti-rejection drugs are not used. However, when Niederkorn feeds the recipient mice skin cells from the donors, only 55 percent of transplants are rejected. And when he also binds the nontoxic B subunit of cholera toxin (CTB) to the skin cells, he's able to lower the rejection rate to just 10 percent (D. Ma et al., British Journal of Ophthalmology, 81:778-84, 1997). Niederkorn hypothesizes that the antigens in the skin cells fed to the donor mice are shuttled across the intestines into aggregations of lymph nodules called Peyer's patches. There the immune system is "reprogrammed . . . in a way that the immune response is down-regulated or inhibited." CTB enhances this process by increasing the transport of the antigens to the Peyer's patches. "Unlike immunosuppressive drugs, this is antigen-specific," Niederkorn comments. "The way we envision doing this [in humans] is, we would go to the tissue bank and obtain skin from the same donor who provided the corneal grafts, take that skin, process it in the laboratory, make a single-cell suspension, put it into gelatin capsules, and give it to patients for 10 straight days." Niederkorn estimates that a section of donor skin the size of two dollar bills would suffice to produce oral tolerance in humans. But if this technique works for corneal transplants, why wouldn't it be useful in preventing the rejection of other transplanted organs? "It's been tried with skin, kidney, and heart in rodents," explains Niederkorn. "The investigators have not used cholera toxin, which I think is an extraordinarily important component of this. And they have seen very modest effects, nothing like what we've seen. But bear in mind that the cornea is transplanted to a unique environment, and the immunology of the eye is very conducive to this. . . .The eye has so many layers of protection against immune assault, that when you put all those layers together . . . and you mix that with antigen feeding, you get an additive effect. At least that's what we think is happening." ANECDOTE: Minnesota's Edward Holland recounts how advances in ophthalmology translated directly into improved vision for one man. The occasional failures of corneal transplants used to be ascribed to rejection in all cases, but Edward J. Holland, a professor of ophthalmology at the University of Minnesota School of Medicine in Minneapolis, has found an interesting class of exceptions. "All donor corneas have a population of epithelial cells from the donor that eventually slough [off]," he explains. "Eventually the patient has to come up with new epithelium that repopulates that donor cornea." These epithelial cells are derived from stem cells at the margins-the "limbus"-of the cornea. "And if the disease affects the limbus . . . and we don't have any stem cells to repopulate the cornea, that transplant is doomed to failure." Holland's solution in such cases is to transplant the corneal limbus from a cadaver, a healthy relative, or-in cases in which only a single eye is affected-the patient's healthy eye. "We can take 50 percent of cells from a healthy donor eye and not see any problems," Holland notes. The technique is called a keratolimbal allograft, and it's clearly of more than academic interest. At the RPB seminar Holland told the story of a man who lost his eyesight in the 1960s as a result of a chemical injury. Four attempts at cornea transplants all eventually failed. "He walked into our clinic a few years ago," recalls Holland, "and he said, 'I haven't been here in 20 years. Is there anything new in ophthalmology that you can offer me?'" Holland first performed a keratolimbal allograft, then three months later the patient received a corneal transplant. "He now has 20/25 vision, he's three years out from surgery, and certainly his whole life has changed." Holland recently reported the results of keratolimbal allograft surgery in 25 eyes of 21 patients (E.J. Holland, Transactions of the American Ophthalmological Society, 94:675-743, 1996). Eighteen of the 25 eyes developed a stable ocular surface, and 15 eyes showed significant improvement in visual acuity. His additional studies are focusing on whether cadavers or living relatives are the best source of donor tissue; the role of immunosuppression in improving the success rate of the procedure; and eye-banking issues, such as how long stem cells can be stored and whether eye banks could actually grow stem cells to meet the demand for this tissue. For Wilson, this work and other studies emphasize that "it's probably the most exciting time in cornea research. The cornea is finally coming into the molecular and cellular age. I think the retina preceded us. It wasn't more than five years ago that people thought of the cornea almost like a piece of plastic." Adds Cavanagh, "The major problems of infection and transplantation are now really amenable to being studied in a molecular way." Robert Finn, a freelance science writer based in Long Beach, Calif., can be reached online at finn@nasw.org.

Advances Help Researchers To See The Cornea More Clearly

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