Subepithelial amyloidosis of the cornea, also called gelatinous drop-like dystrophy (GDD), is a corneal stromal dystrophy with an incomplete autosomal recessive inheritance. 1–3 A genetic abnormality in chromosome 1 was recently discovered. 4 Although there are few reports in the English-language journals, 5–9 most patients with GDD are Japanese. In addition to gradual vision loss, patients with GDD suffer from severe irritating symptoms, such as foreign body sensation, lacrimation, and photophobia, beginning in the first or second decade of life. Although lamellar or penetrating keratoplasty is the sole treatment of visual rehabilitation, eyes that undergo keratoplasty are invariably complicated by recurrence of the disease. Patients therefore require multiple surgeries, which increase the risk of complications, such as cataract, glaucoma, and infection. 1–3
The pathogenesis of GDD remains unknown, but clinical and pathologic findings indicate that the corneal epithelium seems to be involved in the production of amyloid deposits. 2,8,10,11 Pathological examination showed that amyloid deposits are formed in subepithelial lesions. 10–12 Subepithelial glasslike haziness, an early sign of recurrence, develops approximately 8 months after surgery. The period corresponds to the replacement of donor corneal epithelium by recipient epithelium. 12 In addition, bandage soft contact lens wear, which decreases epithelial turnover, was reported to delay postkeratoplasty recurrence. 13
Based on these observations, we hypothesized that host corneal epithelial cells are responsible for amyloid deposition. We then considered that transplantation of allo-limbal stem cells of the cornea may be effective for the treatment of GDD. In the current study, we report nine consecutive eyes with GDD that were treated with limbal stem cell transplantation (LSCT).
PATIENTS AND METHODS
Nine consecutive eyes of seven patients with GDD were treated with LSCT. Demographic profile of the patients are shown in the Table 1. Patients were three males and four females with a mean age of 46.9 years (range, 12–67 years). Four had a family history of GDD, and the parents of two patients were consanguineous. Cases 3 and 4 were brother and sister. Five eyes had a history of keratoplasty, and four of the five eyes had multiple graftings. Preoperative visual acuity ranged from hand motions to 20/600.
The surgical method consisted of excision of the limbal and peripheral corneal tissue by superficial keratectomy. A limbal allograft was prepared from donor corneal rim by trimming the sclera and deep corneal stroma using microscissors. The graft was a two-segment graft (two eyes), C-shaped (three eyes), ring-shaped (three eyes), or a one-piece lamellar graft (one eye). Hyaluronic acid (Healon, Pharmacia-Upjohn, Tokyo, Japan) was used to protect the limbal and corneal epithelium. After lamellar (four eyes) or penetrating (five eyes) keratoplasty, the limbal graft was secured to the recipient's limbal portion using 10-0 nylon sutures. In three of the four eyes that had lamellar keratoplasty, Descemet's membrane was exposed (deep lamellar keratoplasty), and donor corneas whose endothelium was removed were secured on the bed. Extracapsular cataract extraction with implantation of an intraocular lens was performed simultaneously with penetrating keratoplasty in one eye. At the end of surgery, amniotic membrane patching 14 or a bandage soft contact lens was placed over the entire cornea. The amniotic membrane was removed 1 week after surgery, and a contact lens was placed at least several weeks after surgery and continued in two eyes.
After surgery, all patients received epithelial protection therapy and immunosuppression. For epithelial management, artificial tears, hyaluronic acid eyedrops (Hyalein-mini, Santen Pharmaceutical Co., Osaka, Japan), autologous serum eyedrops, and corticosteroids (Solu-Medrol, Pharmacia-Upjohn) were used. All these eyedrops were preservative-free. For immunosuppression, short-term systemic corticosteroids (dexamethasone starting from 8 mg a day and tapered in 2 weeks) and topical and systemic cyclosporin A were used. Systemic cyclosporin A (Sandimmune, Novartis-Pharma, Tokyo, Japan) was used in six eyes starting from 5mg/kg oral administration, with blood through levels maintained at 50 to 100 ng/mL for at least 2 months. Cyclosporin eyedrops were prepared in α-cyclodextrin at a concentration of 0.05%.
To study survival of donor-derived epithelium, fluorescein in situ hybridization (FISH) analysis was performed in some patients who had sex-mismatched LSCT as previously described. 15 Briefly, epithelial cells in the paracentral cornea were obtained, and sex chromosome was hybridized using specific CEP X SpectrumOrange/CEP Y SpectrumGreen Dual Color Probe (Vysis, Downers Grove, IL, U.S.A.). Signals from the probes were visualized by a fluorescence microscope (Nikon, Tokyo, Japan).
For statistical analysis, logarithms of visual acuity were used, and visual acuity evaluated as finger counting and hand motions was converted to 0.004 and 0.002, respectively, for statistical analysis. Preoperative and postoperative visual acuity was compared by using the nonparametric Wilcoxon signed-rank test.
A 44-year-old man first noticed decreased vision and photophobia at 8 years of age. He was diagnosed with GDD at another hospital and underwent lamellar or penetrating keratoplasty a total of seven times. However, recurrence developed several years after each surgery. He also underwent trabeculectomy for secondary glaucoma. His left eye also underwent keratoplasty three times, but he lost vision in his left eye because of fist trauma and subsequent retinal detachment. At his first visit to our hospital, his vision was 20/600 in the right eye and no light perception in his left eye. The right cornea showed dome-like yellowish deposits mainly at the inferonasal quadrant of the central graft and in the peripheral cornea. Marked lipid deposition associated with neovascularization was recognized in the central graft (Fig. 1).
He underwent LSCT combined with penetrating keratoplasty on January 10, 1998. After marking by a trephine of 7.5 mm in diameter, the peripheral cornea was lamellarly dissected. Because corneas with GDD are extremely soft and fragile, care was taken not to perforate the anterior chamber. A central penetrating grafting of 8.0-mm diameter was performed, and the graft was temporally secured by several interrupted 10-0 nylon sutures. Then the limbal graft from the same donor cornea was placed on the recipient's limbal area. The central and limbal grafts were further secured using interrupted and running sutures. Histopathologic examination showed that Congo red–positive material was observed in subepithelial lesions, and the epithelium showed goblet cells (Fig. 2).
After surgery, corneal epithelialization was achieved in 4 days. Mild leakage of the aqueous humor lasted for 2 weeks and resolved by medical treatment. Although oral and topical cyclosporin A and preservative-free dexamethasone eyedrops were used, endothelial rejection developed 5 months after surgery. The limbal graft remained clear. Corneal edema decreased by systemic steroid administration, and the graft maintained its clarity thereafter. At the last examination, vision in the left eye was 20/60 with a hard contact lens. Central and limbal grafts were clear and compact, and central endothelial density was approximately 1,200/mm2 (Fig. 3).
With a mean observation period of 49.4 months (range, 8.9–81.0 months), eight (88.9%) of nine eyes were free of recurrence. One eye had clear and smooth ocular surface epithelia for 6 years, and donor-derived epithelial cells were observed using FISH analysis 4 years after surgery (data not shown). Then irregular epithelium gradually invaded over the corneal surface, which later formed gelatin-like deposits. Corrected visual acuity improved in seven eyes (77.8%), and five eyes obtained more than 20/200.
As early postoperative complications, one eye that underwent lamellar keratoplasty combined with LSCT developed double anterior chamber after a small rupture of Descemet's membrane at the time of surgery. The double chamber was resolved without surgical intervention within 2 weeks. Two other eyes showed marked increases in intraocular pressure. As late complications, glaucoma was seen in five eyes, and three of the cases required surgical intervention. Epithelial and endothelial rejection was recognized in one and two eyes, respectively. No signs of immunologic rejection were found in limbal grafts. All but one eye (case 8) had smooth epithelium on the central graft. One eye (case 6R) developed retinal detachment 45 months after LSCT. Although the retina was successfully repositioned, visual acuity remained at hand motions.
Gelatinous drop-like dystrophy is a severe form of corneal dystrophy with amyloid deposits accumulating mainly in the superficial stroma. Most patients with GDD are Japanese, and prevalence of the disease among the Japanese population is estimated to be 1 in 300,000. 2–4 In the advanced stage, dome-shaped deposits cover the entire corneal surface, and neovascularization with lipid deposition further worsens corneal clarity. Although keratoplasty is the only method for vision restoration, surgery itself is often complicated because of the fragility of the corneal tissue. In addition, subepithelial haziness, which is considered an early sign of recurrence, develops within 1 year of surgery. Recurrence of amyloid deposits follows and necessitates repeated surgery within several years. 12
In the current study, neither subepithelial haziness nor recurrence of deposits was observed in eight of nine patients, with a mean observation period of 4 years. This is markedly different from our previous study, in which all but one (34 of 35) case developed recurrence within 4 years of keratoplasty. 12 This difference is likely to be attributed to difference in surgical approaches used (i.e., transplantation of limbal stem cells).
Limbal stem cell transplantation was first reported by Kenyon and Tseng 16 in 1982, who transplanted limbal tissue from opposite healthy eyes (autograft). Later, Tsai and Tseng 17 reported encouraging results of LSCT using cadaver eyes as donors (allograft). Lines of clinical results of LSCT were reported thereafter, including those for the treatment of aniridia, carcinoma in situ, chemical and thermal burns, Stevens–Johnson syndrome, ocular cicatricial pemphigoid, contact lens–associated epitheliopathy, and chronic keratoconjunctivitis. 18–23 Because we have found donor-derived epithelial cells on the central graft as late as several years after LSCT, 15 stem cells on the allografts continued to supply corneal epithelial cells, preventing abnormal amyloid deposition.
The current study is the first report showing that LSCT is effective in the treatment of corneal dystrophy. Although GDD has been regarded as a corneal stromal dystrophy, efficacy of LSCT observed in the current study supports the hypothesis that the corneal epithelium is responsible for the production of amyloid deposits in GDD.
Although LSCT was effective in supplying healthy corneal epithelia after GDD, intensive postoperative management seemed to be the key for long-term visual improvement. Control of immunologic rejection and intraocular pressure was especially important. It is well known that the limbal area contains numerous Langerhans cells. 24 Therefore, immunologic reaction is likely to be more frequent than lamellar or penetrating keratoplasty. Although many of the patients in this study received oral cyclosporin A therapy, immunologic rejection in the central graft developed in three eyes. We have reported that endothelial rejection in the central graft develops in 36% to 46% eyes that undergo simultaneous central and limbal grafting, 23,25 which is comparable with the rejection rate in the current study.
For the control of intraocular pressure, various factors should be considered. First, we removed perilimbal episcleral tissue, where the efferent routes of aqueous humor exist. Second, we used topical steroid after surgery, which may increase the risk of steroid-related ocular hypertension. Third, the preoperative cornea was irregular and opaque in most eyes, which prevented precise evaluation of their intraocular pressure and optic disk damage. In our long-term follow-up study in eyes with LSCT, 37% of the eyes developed increased intraocular pressure. 23 In the current study, five eyes had visual field changes, and two eyes required surgery for intraocular pressure control. Nonetheless, two eyes lost their central visual field although the cornea remained clear.
In summary, we have shown a novel approach for subepithelial amyloidosis of the cornea, or GDD, using transplantation of limbal stem cells from donor corneas. A marked suppression of recurrence can be obtained using this method. Control of immunologic rejection and glaucoma seems to play a key role in long-term success.
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