Collagen metabolism following corneal laceration in rabbits

We investigated the synthesis and degradation of collagen in lacerated rabbit corneas. The rate of collagen synthesis was measured by the incorporation and hydroxylation of [14C]proline up to 5 weeks after a penetrating laceration. Our results indicate that protein and collagen synthesis is initially reduced during the 24 h period after laceration and then increases in two phases. A moderate increase in protein and collagen synthesis occurs up to day 5 after laceration. Synthesis then decreases to a low level through the 10th day after injury. A second wave of increase in protein and collagen synthesis takes place reaching a new peak of approximately twice the activity as found in control corneas after 5 weeks of healing. Afterwards, the rate of protein and collagen synthesis declines and reaches the basal level after 7 weeks of healing. In a separate set of experiments, rabbit corneas were lacerated and allowed to heal for one or three weeks at which point 100 microCi of [14C]proline was injected into each anterior chamber. The corneas were excised 1 to 17 days later and subjected to collagen analysis. Our data indicates that the degradation of collagen synthesized by the cornea 1 week after laceration followed biphasic kinetics. In the first phase, the half-life of newly-synthesized collagen is 20 days, whereas it is approximately 4 days in the second phase. It is of particular interest that the accelerated degradation of the newly-synthesized [14C]collagen is concomitant with the increased rate of collagen synthesis during the wound healing process. The degradation of collagen synthesized 3 weeks after injury was slower and followed monophasic kinetics having a half-life of 14 days. The degradation of non-collagenous 14C-proteins follow a monophasic kinetics having a half-life of 6-7 days. Polyacrylamide gel electrophoresis of newly-synthesized collagen indicated that the collagen(I) and collagen(V) were the main collagenous components synthesized by the lacerated corneas.     

Transplant of corneal epithelium to rabbit corneal wounds in vivo

Sheets of corneal epithelium removed from 9-mm buttons of adult rabbit corneas using Dispase II were placed on abraded (basement membrane intact) or keratectomized corneas of anesthetized rabbits. Both types of wounds extended from limbus to limbus. The host animals were maintained under deep anesthesia for 3 hr, during which time culture medium was dripped onto the surface of the transplant. A soft contact lens then was placed over the cornea and the eye bandaged shut. Short-term experiments indicated that after 24 hr the transplanted epithelium was adherent to both abraded and keratectomized corneas (n = 4). Hemidesmosomes had formed between basal cells of donor epithelium and denuded host basement membrane, and cytoplasmic blebs had extended from donor epithelium into host keratectomized stroma. Seven transplants to abraded corneas and 17 transplants to keratectomized corneas were followed for longer time periods. Six of the seven transplants to abraded corneas were maintained until termination of the experiment (four at 4 weeks, one at 2 weeks, one at 1 week). Three of the 17 transplants to keratectomized corneas were maintained until termination (one at 4 weeks, one at 2 weeks, and one at 6 days). The remaining 14 sloughed between days 2 and 6. These data indicate that it is feasible to transplant corneal epithelial sheets and that they can be maintained most successfully if the host basement membrane is present.     

Caspase inhibitor z-VAD-FMK inhibits keratocyte apoptosis, but promotes keratocyte necrosis, after corneal epithelial scrape

The purpose of this study was to determine whether the caspase inhibitor z-VAD-FMK could be applied topically prior to epithelial scrape injury to inhibit keratocyte apoptosis. Rabbit corneas were treated with z-VAD-FMK or vehicle alone prior to epithelial scrape injury. Cell fate was analysed at 4 hr after epithelial scrape using quantitative TUNEL assay, propidium iodide staining, and transmission electron microscopy. Less stained anterior stromal keratocytes were detected with the quantitative TUNEL assay in corneas pre-treated with z-VAD-FMK than in corneas pretreated with vehicle at 4 hr after epithelial scrape. This difference appeared to be confirmed by propidium iodide staining of keratocyte nuclei. It was observed that fewer nuclei were stained with propidium iodide in the DMSO vehicle treated corneas compared to the z-VAD-FMK treated corneas. Analysis of corneas with transmission electron microscopy, however, indicated that many anterior stromal keratocytes in corneas pretreated with z-VAD-FMK, but not vehicle, had cell morphologic changes more consistent with necrosis. Although pretreatment of corneas with the caspase inhibitor z-VAD-FMK inhibited keratocyte apoptosis detected with the TUNEL assay, transmission electron microscopy revealed that many anterior stromal keratocytes in z-VAD-FMK-treated corneas instead died by necrosis. Thus, z-VAD-FMK is unlikely to be useful to modulate corneal would healing through inhibition of keratocyte apoptosis induced by epithelial injury. The TUNEL assay should not be used to monitor cell fate without confirmation using analyses that also detect necrosis.     

Keratocyte apoptosis associated with keratoconus.

Keratoconus is an ectatic corneal dystrophy associated with stromal thinning and disruption of Bowman's layer. The purpose of this study was to explore a possible association between keratocyte apoptosis and keratoconus. Keratocyte apoptosis was evaluated in corneas of patients with keratoconus, corneas of patients with stromal dystrophies, and normal donor corneas using the transferase-mediated dUTP-digoxigenin nick and labeling (TUNEL) assay. Keratocyte apoptosis was also studied in keratoconus and normal corneas using transmission electron microscopy. TUNEL-stained keratocytes were detected in 60% of corneas with keratoconus, but only 35% of corneas with stromal dystrophies (P =0.03). The number of TUNEL-positive keratocytes detected in the keratoconus, stromal dystrophy, and normal corneas was 7+/-1 (mean+/-standard error, range 0-20), 2+/-0. 8 (range 0-9), and 0+/-0 (range 0-0) TUNEL-positive cells per section, respectively. The differences between the keratoconus and the stromal dystrophy (P =0.0097) or the normal cornea (P =0.01) groups were statistically significant. The difference between the stromal dystrophy and normal cornea groups was not statistically significant (P =0.45). The stromal dystrophy group was included to account for surgery-associated keratocyte apoptosis. No TUNEL-stained keratocytes were detected in normal corneas. Cell morphologic changes consistent with apoptosis were detected by transmission electron microscopy (TEM) in keratocytes of keratoconus corneas, but not in keratocytes in normal corneas. Chronic keratocyte apoptosis associated with ongoing epithelial injury may link risk factors associated with keratoconus such as chronic eye rubbing, contact lens wear, or atopic eye disease. Similarly, increases that have been detected in several different degradative enzymes in keratoconus corneas could be associated with chronic keratocyte apoptosis and less than perfect control of release of intracellular contents.     

RANK, RANKL, OPG, and M-CSF expression in stromal cells during corneal wound healing

PURPOSE: To examine the influx of monocytes into the cornea after epithelial scrape injury and the expression of chemokines that potentially regulate monocyte phenotype in cultured corneal fibroblasts and keratocytes in situ. METHODS: Monocytes were detected by immunocytochemistry for the monocyte-specific antigen CD11b, in unwounded and epithelial scrape-wounded mouse corneas. The receptor activator of NF-kappa B ligand (RANKL), osteoprotegerin (OPG), and monocyte chemotactic and stimulating factor (M-CSF) mRNAs were detected in cultured mouse stromal fibroblasts by RT-PCR and RNase protection assay. RANKL, OPG, and M-CSF proteins were detected in cultured mouse stromal fibroblasts by immunoprecipitation and Western blot analysis. RANKL, RANK, M-CSF, and OPG proteins were detected in unwounded and wounded mouse corneas by immunocytochemistry. Chimeric mice with green fluorescent protein-labeled bone marrow-derived cells underwent corneal scrape injury and were monitored by fluorescence microscopy and immunocytochemistry. RESULTS: A small number of cells expressing the monocyte-specific CD11b antigen were detected in the stromas of unwounded mouse corneas. A larger number of CD11b-positive cells was detected in the stroma at 24 or 48 hours after epithelial scraping injury. Experiments with chimeric mice with fluorescent green protein-labeled, bone marrow-derived cells demonstrated conclusively the origin of these CD11b(+) cells. RANKL, OPG, and M-CSF mRNAs and proteins were detected in cultured mouse stromal fibroblasts. RANKL, M-CSF, and OPG proteins were detected in unwounded corneas, but were expressed at higher levels in stromal cells during the 24- to 48-hour interval after epithelial scrape injury. RANK was detected in stromal cells presumed to be monocytes at 24 and 48 hours after epithelial injury. CONCLUSIONS: Cells expressing the CD11b monocyte-specific antigen appear in the corneal stroma in high numbers by 24 hours after epithelial injury and persist beyond 10 days after wounding. Cultured corneal fibroblasts and keratocytes in situ express RANKL, OPG, and M-CSF cytokines involved in regulating osteoclast differentiation from monocytes in bone. Cells expressing RANK were detected in the stroma at 24 and 48 hours after epithelial injury. The cytokine systems that regulate monocyte transition to osteoclast in bone are upregulated in the cornea in response to epithelial injury and may participate in regulating monocyte phenotype during corneal stromal wound healing.     

Therapeutic action of meliacine,a plant-derived antiviral,on HSV-induced ocular disease in mice

Ocular herpes simplex virus type-1 (HSV-1) infections remain an important cause of corneal disease which may result in a loss of vision. Meliacine (MA), an antiviral activity present in crude leaf extracts of Melia azedarach L. that inhibits HSV-1 multiplication in vitro, was studied in a murine herpetic stromal keratitis experimental model. Adult Balb/c mice were inoculated with HSV-1 at their corneas after abrasion. MA was administered topically three times a day for 3 consecutive days, beginning at 24 and 96 hr after infection. Infected animals treated or not with MA were monitored for the development of ocular disease by a binocular microscope for 16 days. MA significantly reduced the incidence and the severity of blepharitis, neovascularization and stromal keratitis with respect to untreated infected mice, regardless the schedule of treatment assayed. Histological examination of corneas from MA-treated animals revealed no tissue damage, whereas corneal samples from untreated infected mice showed inflammation, vascularization and necrosis. In uninfected mice treated with MA, we found no evidence of corneal damage and histopathological studies showed no changes in the corneas of these mice. Treatment with MA at 24 hours post-infection (h.p.i.) reduced viral multiplication in the eye by 1-1.5 orders of magnitude. Studies on latency revealed that MA sligthly affected the establishment of a latent infection. Thus, MA proved to exert an antiviral action on the development of herpetic stromal keratitis when supplied by post-treatment. Unexpectedly, treatment with MA after 96h.p.i prevented ocular disease, suggesting an in vivo immunomodulating activity of MA.     

Acid proteases and histologic correlations in experimental ulceration in vitamin A deficient rabbit corneas

Although xerophthalmia due to severe vitamin A deficiency is the leading cause of childhood blindness in the underdeveloped countries, little is known about the proteases (other than collagenase) that are involved in the degradative mechanism. The degree of cellular autolysis and stromal degradation observed histologically in early stages of xerophthalmia and in ulcerating corneas in vitamin A deficient rabbits in this study were, in general, proportional to the levels of the proteases studied. The only major histologic and ultrastructural alteration observed in early xerophthalmic corneas was autolysis of superficial epithelial and stromal cells. In contrast, in the ulcerating corneas the stroma was infiltrated heavily with inflammatory cells and extensive stromal degradation was observed in the central necrotic region of the lesions. Maximal proteolytic activity toward hemoglobin was observed at pH 3.3 for corneal extracts from normal (N) and pair-fed control (C) rabbits and rabbits with early xerophthalmia (X) and ulcerating xerophthalmia (U) corneas. This activity was a cathepsin D-like enzyme per cornea that had a ratio of 1:1:3:16 in the N, C, X, and U corneas. The ratio of cathepsin B-like activity per cornea for N, C, X, and U corneas was 1:2:2:10.     

Collagen ultrastructural changes during stromal wound healing in organ cultured bovine corneas

Corneal collagen ultrastructural changes occur during the healing process. The present study was designed to compare collagen ultrastructural changes after trephine wounding or flap creation. Bovine corneas were injured and maintained in organ culture for up to 4 weeks. Samples were removed from culture at 0, 1, 2, 3 and 4 weeks and snap frozen in liquid N₂. X-ray scattering was used to measure changes in collagen interfibrillar spacing, intermolecular spacing and fibrillar diameter. Some samples were fixed in 10% Neutral Buffered Formalin solution and wax embedded for immunohistochemistry to monitor myofibroblast differentiation in corneal flaps. Swelling effects (i.e. changes in interfibrillar spacing) were more severe in trephined corneas than in those with stromal flaps. Collagen fibrillar diameter remained normal in the periphery of injured corneas, but increased significantly in areas within and around the wound in trephined samples and in the epithelial incision site in corneal flaps. Intermolecular spacing was unchanged in all samples. In the flaps, αSMA expression was only detected in an area adjacent to the epithelial plug, and cell numbers gradually increased during the culture. We conclude that stromal swelling is more rapid for trephine-wounded corneas than in stromal flaps, indicating that the intensity of the corneal healing response depends on the type of injury.     

Regulation of endotoxin-induced keratitis by PECAM-1, MIP-2, and toll-like receptor 4

PURPOSE: Bacterial lipopolysaccharide (LPS, endotoxin) is a potent stimulator of inflammatory responses and is likely to contribute to microbial keratitis and to the pathogenesis of sterile corneal ulcers. The purpose of the present study was to identify specific mediators of endotoxin-induced keratitis. METHODS: The corneal epithelium of BALB/c, C3H/HeJ, and C3H/HeN mice was abraded, and Pseudomonas aeruginosa endotoxin (10 microg in 1 microL) was added. Stromal thickness and haze were measured by in vivo scanning confocal microscopy, and neutrophil recruitment determined by immunohistochemistry. RESULTS: Pseudomonas endotoxin induced a significant increase in stromal thickness and haze compared with untreated control corneas at each time point examined, and the severity coincided with neutrophil infiltration into the corneal stroma. Furthermore, systemic depletion of neutrophils completely abrogated endotoxin-induced increases in stromal thickness and haze, indicating an essential role for these cells. Expression of platelet endothelial cell adhesion molecule (PECAM)-1 on vascular endothelium and production of macrophage inflammatory protein (MIP)-2 in the corneal stroma were also significantly elevated after exposure to endotoxin, and antibody blockade inhibited neutrophil recruitment to the cornea and abrogated endotoxin-induced increases in stromal thickness and haze. In LPS-hyporesponsive C3H/HeJ mice, PECAM-1 and MIP-2 were not upregulated after exposure to endotoxin, and endotoxin-induced keratitis did not develop in these mice. CONCLUSIONS: These findings demonstrate that endotoxin-induced keratitis is regulated by toll-like receptor-4 (TLR4)-dependent expression of PECAM-1 and MIP-2, which are essential for recruitment of neutrophils to this site and for development of endotoxin-induced stromal disease.     

20%乙醇处理兔角膜后上皮增生和细胞凋亡的研究

目的探讨准分子激光角膜上皮瓣下磨镶术(LASEK)中采用20%乙醇浸润兔角膜40s后角膜上皮增生和角膜细胞凋亡情况与机械刮除角膜上皮后的异同。方法实验组42只新西兰大白兔,用直径为8mm的LASEK专用角膜上皮刀切割角膜上皮,20%的乙醇浸润单眼40s,机械刮除对侧眼中央8mm直径的角膜上皮,随机分7组,于术后0、4h,1、3、5、8、30d取材;6只兔眼为空白对照。角膜冰冻切片,行Ki67免疫组化检查和TUNEL检测,计数角膜中央前基质细胞。结果乙醇浸润后5d中央角膜上皮增生达峰值,术后1d周边角膜上皮增生达峰值;术后4h上皮刀口下方局限的角膜基质细胞TUNEL染色阳性,数量最多;各组角膜中央前基质细胞计数和空白对照比差异无统计学意义(P=0.68)。机械刮除角膜上皮后3d周边角膜上皮增生达峰值,其高于乙醇浸润后角膜上皮的增生峰值;术后4h可见大量中央前基质细胞TUNEL阳性;术后1d中央前基质细胞数量最少(P<0.05)。结论与机械刮除角膜上皮相比,20%乙醇浸润40s对角膜损伤轻,恢复快,乙醇浸润后的角膜上皮对基质细胞有保护作用。     

Neonatal corneal stromal development in the normal and lumican-deficient mouse

PURPOSE: The purpose of this study was to characterize temporally stromal growth and transparency in lumican-deficient and normal neonatal mice. METHODS: Lumican-deficient mice and CD1 wild-type mice were evaluated by in vivo confocal microscopy through-focusing (CMTF) to quantify stromal and epithelial thickness and corneal light-scattering and by laser scanning CM to determine density of keratocytes from 1 day to 12 weeks after birth. RESULTS: CD1 corneas showed a rapid loss of light-scattering, decreasing by 50% from day 1 to day 12, that paralleled a 60% decrease in density of keratocytes. By contrast, the stroma demonstrated a marked swelling from day 8 to day 12, followed by thinning at day 14. Compared to corneas from CD1 mice, lumican-deficient corneas showed significantly increased (P < 0.05) light-scattering beginning at week 3 that remained elevated above wild-type levels for the duration of the study. Stromal development was also markedly altered, with thinning detected at week 3, followed by no detectable stromal growth for the duration of the study. Density of keratocytes was significantly increased, but the total cell number was similar compared with that in the wild-type cornea, suggesting no effect on keratocyte differentiation. CONCLUSIONS: Development of normal neonatal corneal transparency appears related to changes in density of keratocytes. The stroma, however, undergoes a marked swelling and thinning at the time of eyelid opening (days 8-14). In the lumican-deficient mouse, stromal swelling is abolished, indicating that this critical phase in stromal development is lumican dependent and essential for normal stromal growth and maintenance of stromal transparency.     

Contribution of virus and immune factors to herpes simplex virus type I-induced corneal pathology

In vivo T-lymphocyte subpopulation depletion techniques were used to identify the roles of L3T4+ (CD4) and Lyt-2+ (CD8) T-lymphocytes in the pathogenesis of corneal stromal disease induced by two different strains of Herpes simplex virus type 1 (HSV-1). Histologic examination of infected corneas revealed significant differences in the composition of the inflammatory corneal infiltrates induced by the RE and KOS strains of HSV-1. The RE strain induced a predominantly polymorphonuclear leukocyte (PMN) infiltrate, which began approximately 1 week after infection and progressed through day 21. Depletion of CD4 cells before corneal infection with RE HSV-1 greatly reduced the incidence and severity of corneal disease; depletion of CD8 cells had no effect. The strain KOS HSV-1 induced an early PMN infiltrate that became predominantly mononuclear by day 21. Depletion of CD4 cells did not change the incidence or severity of KOS HSV-1-induced corneal stromal disease. The corneal lesions of these mice contained numerous CD8 cells. Depletion of CD8 cells before KOS HSV-1 infection of the cornea moderately reduced the incidence of stromal disease. However, in CD8-depleted mice with the disease, PMNs were the most prevalent infiltrating cells, and the disease appeared identical to that seen in RE HSV-1 infected corneas. Simultaneous depletion of CD4 and CD8 cells before KOS HSV-1 infection eliminated stromal disease. However, when T-cell depletion was discontinued in these mice, stromal disease developed in concert with the appearance of T-cells in the lymphoid organs and corneas. Thus, T-lymphocytes are a necessary component of HSV-1 corneal stromal disease. These results further suggest that RE HSV-1 preferentially activates CD4 cells in the cornea, and KOS HSV-1 preferentially activates CD8 cells in the cornea.     

Influence of the corneal epithelium on the efficacy of topical antifungal agents

A model of deep stromal Candida albicans infection was established by injecting 25 microliters of a suspension containing 5 X 10(9) colony forming units/ml of the yeast into corneas of pigmented rabbits. In this model, the infection lasts for more than 8 days. Using quantitative techniques, the authors compared the efficacy of six topical antifungal agents in the presence of an intact epithelium and in corneas debrided of epithelium. In corneas debrided on a daily basis, the polyenes (amphotericin B 0.15% and 0.075% and natamycin 5%) exhibited a significant antifungal effect. When the epithelium was left intact, 5% natamycin and 0.075% amphotericin B were without effect, while the efficacy of the 0.15% preparation of amphotericin B was much reduced. Removal of the epithelium appeared to affect adversely the efficacy of flucytosine. The imidazoles were not efficacious in this model.     

Participation of T lymphocyte in corneal edema in the early stage of herpetic stromal keratitis

The participation of T lymphocytes in the immunopathogenesis of corneal opacity in herpetic stromal keratitis was investigated. In BALB/c mice infected intracorneally with herpes simplex virus type 1, corneal opacity was manifested 10 days after infection, while in athymic mice corneas remained almost clear. Histologically, all opaque corneas revealed stromal edema accompanied by the diffuse presence of polymorphonuclear cells and lymphocytes. When complement (C')-treated immune spleen cells were adoptively transferred into athymic mice 6 or 72 h after corneal infection, stromal keratitis with mild opacity was observed 10 days after transfer. The athymic mice given anti-Thy 1.2+C'-treated immune spleen cells failed to develop corneal opacity. The difference, as revealed by light and electron microscopy, was the presence or absence of lymphocytic infiltration and edema in the posterior third layers of the stroma and endothelial lesions. The endothelium was infiltrated by lymphocytes or macrophages and showed various stages of destruction. The main cause of corneal opacity in the early stage of herpetic stromal keratitis is thought to be stromal edema due to an adverse effect on the endothelium by immune T lymphocytes.     

Recovery of herpes simplex virus from ocular tissues of latently infected inbred mice

Evidence for latent infection of ocular tissues following topical corneal inoculation with herpes simplex virus type 1 (HSV) was sought in three strains of inbred mice that differ in susceptibility to HSV stromal keratitis. Corneas of BALB/c, C57BL/6, and DBA/2 mice were inoculated topically with HSV. At 6-8 weeks after inoculation, when no active ocular infection was present, minced whole eyes and trigeminal ganglia were assayed for latent virus. Virus was recovered by explantation from minced eyes of all three strains (DBA/2 = 20%; BALB/c = 17%; C57BL/6 = 7%). In order to determine which ocular structures harbored virus, corneas, retinas and choroid-sclera were cultivated separately. Virus was activated from corneas of DBA/2 and BALB/c mice, but not from corneas of C57BL/6 mice. These findings suggest that HSV is capable of establishing latent infection in ocular tissue of inbred mice and that the rate of establishment of latency is under host genetic control. Since neural cell bodies are not present in the cornea, the data suggest that latency is established in cells other than neurons.     

Alpha-1 proteinase inhibitor levels in keratoconus

The levels of alpha 1-proteinase inhibitor (alpha 1-antitrypsin) in keratoconus, normal human, and other diseased corneas were examined. Using an immunoperoxidase technique, the presence of this inhibitor was demonstrated in the epithelium, stroma and endothelium of all corneal sections. Compared with normal human controls, the staining intensity in the epithelium and stromal lamellae of keratoconus corneas was markedly reduced. Such a reduction was not seen in either scarred or other diseased corneas. Extracts of keratoconus and normal human corneas were subsequently analyzed for alpha 1-proteinase inhibitor by a dot blot assay using a monoclonal antibody against the inhibitor and a 125I-labelled secondary antibody. In agreement with the immunohistochemical findings, the alpha 1-proteinase inhibitor level found in the epithelium of keratoconus corneas was approximately one-fourth of that found in normal human controls. In addition, the stromal extracts of keratoconus corneas contained about one-sixth the inhibitor level of that in normal human extracts. These results lend further support to the hypothesis that degradation processes may be aberrant in keratoconus.     

Role of corneal collagen fibrils in corneal disorders and related pathological conditions

The cornea is a soft tissue located at the front of the eye with the principal function of transmitting and refracting light rays to precisely sense visual information. Corneal shape, refraction, and stromal stiffness are to a large part determined by corneal fibrils, the arrangements of which define the corneal cells and their functional behaviour. However, the modality and alignment of native corneal collagen lamellae are altered in various corneal pathological states such as infection, injury, keratoconus, corneal scar formation, and keratoprosthesis. Furthermore, corneal recuperation after corneal pathological change is dependent on the balance of corneal collagen degradation and contraction. A thorough understanding of the characteristics of corneal collagen is thus necessary to develop viable therapies using the outcome of strategies using engineered corneas. In this review, we discuss the composition and distribution of corneal collagens as well as their degradation and contraction, and address the current status of corneal tissue engineering and the progress of corneal cross-linking.     

Use of amniotic membrane graft and corneal transplantation in a patient with bilateral keratomalacia induced by uncontrolled phenylketonuria

PURPOSE: To report a patient with a rare complication of bilateral keratomalacia induced by uncontrolled phenylketonuria (PKU) that was successfully treated with amniotic membrane transplantation in 1 eye and penetrating keratoplasty in the second eye. METHODS: Case report and literature review. RESULTS: A 9-month-old girl with uncontrolled PKU was referred to our clinic because of bilateral keratomalacia. Slit-lamp examination of the right eye revealed 2 large corneal erosions with stromal thinning on the nasal and inferior regions of the right cornea. Left eye examination revealed a large area of melting involving two thirds of the cornea with corneal perforation and iris bulging on the temporal side and no anterior chamber. She underwent amniotic membrane transplantation on her right cornea and penetrating keratoplasty on her left cornea. Treatment after surgery included antibiotic and steroid eye drops and a special diet regimen with partial phenylalanine intake. Examination under anesthesia 4 months after surgery revealed intact cornea in her right eye and a clear corneal graft with a deep anterior chamber on her left eye. Intraocular pressure was normal in both eyes. CONCLUSIONS: Bilateral keratomalacia, although a rare ophthalmic manifestation of PKU, can cause a severe corneal injury that may threaten the eyeball integrity. Surgical treatments with amniotic membrane graft and corneal transplantation, along with the appropriate diet treatment, were found to be effective procedures, yielding rapid healing of the corneal surface.