Effects of Switching from Timolol to Brimonidine in Prostaglandin Analog and Timolol Combination Therapy.

PURPOSE: To assess the efficacy and safety of switching from timolol 0.5% to brimonidine 0.1% in patients with glaucoma treated with a prostaglandin analog (PGA) and timolol combination. METHODS: This prospective, open-label multicenter study enrolled patients with open-angle glaucoma or ocular hypertension who were being treated with a PGA and timolol. After baseline measurements, timolol was switched to brimonidine 0.1%, and patients were followed for 12 weeks. Patients visited at 4 and 12 weeks after switching. The main outcome measure was the change in intraocular pressure (IOP). For safety evaluations, hyperemia, formation of conjunctival follicles, superficial punctate keratopathy as a local side effect, blood pressure (BP), and heart rate (HR) were evaluated. RESULTS: One hundred seven patients participated in this study. Among them, 103 patients completed the study. The IOP values at baseline, 4 weeks, and 12 weeks after the transition were 15.7 ± 2.7, 14.3 ± 2.8, and 14.0 ± 2.8 mmHg, respectively. IOP was significantly reduced at 4 and 12 weeks compared with baseline (p<0.001). There were no significant changes in hyperemia or follicle formation. The superficial punctate keratopathy score was significantly reduced at 12 weeks compared with baseline (p<0.05). Systolic and diastolic BP values were significantly reduced and HR significantly increased after switching (p<0.05). CONCLUSION: Switching from timolol 0.5% to brimonidine 0.1% may not change IOP in the combination use of timolol 0.5% and a PGA and was well tolerated by patients without severe ocular or systemic side effects.

New graft inserter for descemet stripping automated endothelial keratoplasty.

PURPOSE: The aim of this study was to introduce a newly developed graft inserter for Descemet stripping automated endothelial keratoplasty, which can be readily used with 1 hand, and to examine endothelial cell loss (ECL) after graft insertion using this new inserter in an ex vivo rabbit model. METHODS: Rabbit corneal grafts (∼150 µm) were prepared by using a femtosecond laser. The grafts were inserted into an artificial anterior chamber by the pull-through technique using a Busin glide or the push-out technique using our new inserter. ECL was evaluated by means of trypan blue and alizarin red S staining. Grafts that were not injected served as a control group. RESULTS: The mean percentages of ECL area in the control, Busin glide, and new inserter groups were 18.7 ± 1.79%, 25.4 ± 2.32%, and 24.7 ± 1.56%, respectively. The mean percentages of the ECL area in both the Busin glide group and new inserter groups were significantly greater than that in the control group (P = 0.001). There was no statistically significant difference between the Busin glide group and the new inserter group. CONCLUSIONS: Our new inserter may provide a favorable option for donor tissue insertion in Descemet stripping automated endothelial keratoplasty procedures.

Caffeine increases tear volume depending on polymorphisms within the adenosine A2a receptor gene and cytochrome P450 1A2.

PURPOSE: The primary aim of the present study was to examine the effect of caffeine on tear volume. The secondary aim was to investigate the relation between caffeine-induced changes in tear volume and polymorphisms in ADORA2A and CYP1A2. DESIGN: Double-masked, placebo-controlled, crossover study. PARTICIPANTS: Seventy-eight healthy volunteers were recruited for the study. METHODS: Subjects participated in 2 sessions in which they received capsules containing either placebo or caffeine. The caffeine capsules were given to the subjects to keep the caffeine volume per body weight within 5 to 7 mg/kg. After caffeine intake, tear meniscus height (TMH) was measured. Subjects provided a blood sample for genotyping. MAIN OUTCOME MEASURES: Tear meniscus height, single nucleotide polymorphism. RESULTS: The tear volume increased after caffeine consumption. The net increase in TMH was 0.08 mm (95% confidence interval, 0.05-0.10) greater when participants were given caffeine than when given placebo (P<0.0001). In ADORA2A, the difference in the net increase in TMH for participants who were heterozygous at rs5751876 and rs2298383 was 0.07 mm (P = 0.001) and who were minor homozygous was 0.08 mm (P = 0.007). In CYP1A2, the net increase in TMH for participants who were minor homozygous at rs2472304 was lower than for those who were major homozygous; the difference was 0.06 mm (P = 0.039). CONCLUSIONS: Caffeine intake increases tear volume and polymorphisms within ADORA2A, and CYP1A2 is associated with the tear increase after caffeine intake. Genetic polymorphisms had a significant effect on tear meniscus that was of limited clinical significance.

Effect of trabeculectomy on ocular and corneal higher order aberrations.

PURPOSE: To examine the effect of trabeculectomy on ocular and corneal higher order wavefront aberrations. METHODS: Trabeculectomy with a limbal-based conjunctival flap was performed in 13 patients (13 eyes). Ocular and corneal higher order aberrations were measured using a wavefront analyzer before and after surgery. The higher order aberrations for a 4-mm pupil diameter were expanded into Zernike's polynomials. Coma aberration, spherical aberration, and total higher order aberrations were evaluated as root mean square values. RESULTS: Mean intraocular pressure was significantly reduced from 15.5 ± 3.1 mmHg preoperatively to 7.5 ± 3.5 and 7.2 ± 4.4 mmHg at 1 and 3 months after surgery, respectively. The mean spherical equivalent refractive error was not changed by the surgery. Mean vector powers for surgically induced refractive changes at 1 and 3 months postoperatively were 1.29 ± 0.69 and 1.30 ± 0.89 diopters, respectively. Corneal coma-like, spherical-like, and total higher order aberrations were not significantly changed by surgery. Trabeculectomy significantly increased ocular coma-like (P = 0.0021) and total (P = 0.0019) higher-order aberrations at 1 month, but not at 3 months postoperatively. CONCLUSION: Ocular coma-like and total aberrations were significantly increased at 1 month after trabeculectomy with a limbal-based conjunctival flap, but they had returned to normal levels by 3 months.

Identification of Schlemm's canal and its surrounding tissues by anterior segment fourier domain optical coherence tomography.

PURPOSE: To identify Schlemm's canal (SC) and trabecular meshwork (TM) by anterior segment Fourier-domain optical coherence tomography (AS-FD-OCT) with histologic confirmation in enucleated human eyes and to quantitatively evaluate SC and TM in living human eyes. METHODS: In enucleated human eyes, the imaging of the anterior chamber angle by AS-FD-OCT was performed before and after surgical expansion of SC with an injection of a viscoelastic material, followed by histologic examination. In 60 living human eyes, the agreement of identification of SC between examiners was evaluated with the Cohen's κ values, and the lengths of SC and TM and the area of TM were measured on temporal and nasal sections of the AS-FD-OCT images. RESULTS: In enucleated human eyes, SC was observed to be a thin, linear, lucent space in the AS-FD-OCT image obtained with the high-definition raster scan protocol, but not in those obtained with the bi-angle radial scan protocol. This space was enlarged after the SC expansion. In the histologic study, the SC was confirmed to be in the same position as in the AS-FD-OCT images. The κ values of observable SC in living human subjects were 0.92 or higher. The axial length of the SC averaged 347.2 ± 42.3 µm, TM length 466.9 ± 60.7 µm, and TM area 0.0671 ± 0.0058 mm². These measurements showed sufficient repeatability and reproducibility. CONCLUSIONS: Using the high-definition images of the AS-FD-OCT, SC and its surrounding tissues were successfully observed in most of the living eyes and were quantitatively evaluated in a noninvasive manner.

Laser treatment of giant iris cyst with nanophthalmos.

We report a case of a giant iris cyst treated with neodymium-doped yttrium aluminum garnet laser cystotomy. A 33-year-old woman presented with nanophthalmos and ocular hypertension secondary to a giant iris cyst after congenital cataract surgery in the left eye. Examination with a rotating Scheimpflug camera and anterior-segment optical coherence tomography revealed a large iris cyst surrounding the pupillary region. She underwent laser iridocystotomy and irrigation of the anterior chamber. No recurrence of the iris cyst has been noted after 18 months and the IOP is normal. The cyst was treated successfully by laser cystotomy without other ocular tissue damage. Examination with a rotating Scheimpflug camera and anterior-segment optical coherence tomography proved to be simple and quick methods for assessing the iris cyst.

Effect of latanoprost on the expression of matrix metalloproteinases and tissue inhibitor of metalloproteinase 1 on the ocular surface.

OBJECTIVE: To investigate the effect of topical latanoprost on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase 1 (TIMP-1) on the ocular surface. METHODS: Tears were collected from 39 patients with glaucoma who used latanoprost, 0.005%, eyedrops (Xalatan) and 28 healthy volunteers. The MMP-9 concentration was measured. Conjunctival epithelial cells were collected from 10 eyes of 10 patients before and 1 to 3 months after starting to take topical latanoprost, 0.005%, and MMP-1, MMP-9, and TIMP-1 messenger RNA (mRNA) expression was analyzed. Both eyes of 48 mice were treated once a day with latanoprost, 0.005%, timolol gel, 0.5%, eyedrops, vehicle of Xalatan, or phosphate-buffered saline, and MMP-9 and TIMP-1 mRNA expression was analyzed. RESULTS: The median MMP-9 concentration in latanoprost-treated cases was 91.2 ng/mL (in controls, 19.7 ng/mL; P < .001). In latanoprost-treated cases, the relative ratio of MMP-9 to glyceraldehyde 3-phosphate dehydrogenase mRNA was significantly increased from 6.42 to 21.3 (P = .04, paired t test) and the relative amount of TIMP-1 was significantly decreased from 154 to 105 (P = .009). The relative amount of MMP-1 to GAPDH mRNA before and after latanoprost use was not significantly different (P = .16). In mice, MMP-9 expression was increased and TIMP-1 expression was decreased on the ocular surface at 8 weeks after latanoprost use. CONCLUSION: The topical use of latanoprost increases MMP-1 and MMP-9 and decreases TIMP-1 on the ocular surface. CLINICAL RELEVANCE: The use of topical latanoprost might not be recommended in patients with keratoconus or after laser-assisted in situ keratomileusis.

Extended long-term results of penetrating keratoplasty for keratoconus.

PURPOSE: To investigate the extended long-term results of penetrating keratoplasty (PK) for keratoconus. METHODS: The subjects were all patients with clinical keratoconus who underwent initial PK at University of Tokyo from 1971 to 1990 and whose medical records were available. Data were retrieved from the medical records. Kaplan-Meier survival analysis was performed to estimate the cumulative probability of immune reaction-free grafts and graft survival. RESULTS: One hundred twenty-five eyes in 125 patients met the entry criteria. The average age at the time of PK was 25.0 +/- 8.9 years. With an average follow-up period of 15.3 +/- 9.7 years, 12 eyes (9.6%) experienced rejection and 5 eyes (4.0%) experienced graft failure. Average best-corrected visual acuity in logarithm of the minimum angle of resolution at preoperation, 10, 20, and 25 years after surgery was 1.54 +/- 0.68, 0.06 +/- 0.22, 0.03 +/- 0.17, and 0.14 +/- 0.42, respectively. The cumulative probability of graft survival at 10, 20, and 25 years after PK was 98.8%, 97.0%, and 93.2%, respectively. CONCLUSIONS: Recovery of visual acuity was good after PK for keratoconus in an extended follow-up. The graft survival rate, however, gradually decreased 20 years after PK.

Recent causes of subconjunctival hemorrhage.

PURPOSE: To assess the relationship between the incidence of subconjunctival hemorrhage (SCH) and age during each season of the year, and to examine recent risk factors for SCH. METHODS: A total of 161 patients with SCH aged 1-94 years were involved in the study. The age, gender, medical history and ocular history were determined for all subjects. RESULTS: The peak age of onset of SCH was between 61 and 70 years. Fourteen patients (77.7%) had trauma or contact-lens-induced injury, and 4 patients (22.3%) among the younger patients aged 0-40 years had an unknown etiology. Among the older patients aged 61-94 years, the chief risk factor for SCH was hypertension (47.5%), followed by unknown etiology (39.4%) and then diabetes (13.1%). CONCLUSION: These results suggest that the major current risk factors for SCH are trauma and contact-lens-induced injury in younger patients, while hypertension is the main factor in older patients. The incidence of acute hemorrhagic conjunctivitis was decreased.

Location and extent of subconjunctival hemorrhage.

PURPOSE: Subconjunctival hemorrhage (SCH) is a relatively frequent disease; however, there have been no reports about its location and extent. We examined its location and extent. METHODS: A total of 151 patients with SCH aged 2-94 years were studied. The conjunctiva was divided into 8 equal areas. The age, gender, medical history, ocular history and site of hemorrhage were determined for all subjects. RESULTS: The number of areas involved by SCH showed an age-related increase. Traumatic SCH had a smaller extent compared with SCH related to hypertension, diabetes and hyperlipidemia, or idiopathic SCH. Overall, SCH was significantly more common in the inferior areas than the superior areas (55.3% vs. 25.0%, p < 0.000001). In patients with SCH secondary to trauma or diabetes, however, the temporal areas were affected more often than the nasal areas (61.5% vs. 30.8% and 73.3% vs. 20.0%, respectively). CONCLUSION: SCH showed an age-related increase in extent and was predominant in the inferior areas. However, traumatic SCH was usually detected as localized hemorrhage in the temporal areas.

Relationship between myopia and allergen-specific serum IgE levels in patients with allergic conjunctivitis.

PURPOSE: Various exogenous allergens can cause allergic conjunctivitis, whereas refractive errors are also related (as an endogenous factor) to the pathogenesis of allergic conjunctivitis. However, little is known about the interaction between exogenous and endogenous factors in patients with allergic conjunctivitis. We investigated the relationship between refractive errors and exogenous allergens in patients with allergic conjunctivitis. METHODS: Sixty patients with allergic conjunctivitis and 60 healthy subjects (non-allergy patients) were enrolled. Refraction was carried out in all subjects. In addition, total immunoglobulin E (IgE) and specific IgE levels for 12 inhaled allergens were measured by the capsulated hydrolic carrier polymer system. RESULTS: The patients who were positive for-specific IgE to indoor allergens had higher myopia than those who were negative, including those positive for house dust IgE (-3.66 +/- 2.95 vs. -1.05 +/- 3.39, P = 0.0015), Dermatophagoides pteronyssinus (D. pteronyssinus) IgE (-3.50 +/- 2.91 vs. -1.01 +/- 3.46, P = 0.0021), and acarus IgE (-3.45 +/- 2.21 vs. -1.51 +/- 3.73, P = 0.0087), whereas antibody-positive and antibody-negative patients for outdoor allergens showed no significant differences of refraction. Refractions in indoor group (-3.58 +/- 3.42) and indoor/outdoor (-3.70 +/- 2.64) group were higher than those in outdoor group (-0.84 +/- 2.30, P = 0.0047 and P = 0.0032) and the non-allergy group (-1.30 +/- 1.48, P = 0.0082 and P = 0.0054) [-refraction] was significantly correlated with total IgE (r = 0.333, P = 0.0093), house dust (r = 0.355, P = 0.0054) and D. pteronyssinus (r = 0.379, P = 0.0028), while no correlation between refractive error and outdoor allergens was proven. CONCLUSION: These results suggest that specific IgE levels for indoor allergens, such as house dust, might be associated with refractive errors.

Descemet stripping automated endothelial keratoplasty using cultured corneal endothelial cells in a rabbit model.

OBJECTIVE: To investigate the feasibility of Descemet stripping automated endothelial keratoplasty (DSAEK) using cultured human corneal endothelial cells (HCECs) in an animal model. METHODS: Descemet stripping automated endothelial keratoplasty grafts were produced by seeding cultured HCEC suspensions onto human corneal stromal discs. Three insertion techniques were assessed in an ex vivo model. The feasibility of DSAEK grafts with cultured HCECs was examined in a rabbit model. Rabbits received stromal disc transplants with cultured HCECs (c-DSAEK) or without HCECs (controls). RESULTS: The HCECs on the DSAEK grafts had a consistent size and polygonal shape. Mean (SD) percentage of cell loss in the taco-folding group (38.7% [5.2%]) was significantly greater than that in the Busin glide (11.6% [1.5%]; P = .001) and lens glide (18.0% [5.4%]; P = .007) groups. Corneal transparency gradually recovered in the c-DSAEK group, whereas edema persisted for up to 28 days in controls. Histologic examination after surgery revealed donor HCECs covering the posterior surface of the graft in the c-DSAEK group. CONCLUSIONS: Further enhancements of the efficacy and safety of DSAEK using cultured HCECs will make this a clinically feasible alternative therapy for corneal endothelial dysfunction. CLINICAL RELEVANCE: Descemet stripping automated endothelial keratoplasty using cultured HCECs may be a novel therapeutic approach to treat corneal endothelial dysfunction.

Severe hyperopic shift and irregular astigmatism after radial keratotomy.

OBJECTIVES: To report a case of acute high hyperopia occurring after radial keratotomy and evaluate the extent of corneal wavefront aberration with a Hartmann-Shack aberrometer. METHODS: A 45-year-old man had undergone bilateral radial keratotomy with four incisions for myopia 15 years earlier. The patient developed significant hyperopia and irregular corneal astigmatism in the left eye after the operation, reducing his best-corrected visual acuity. RESULTS: The best-corrected visual acuity was 20/20 (with refraction of +0.25-1.50 diopters [D] x 169) in the right eye and 10/20 (with refraction of +9.5D-3.00D x 50) in the left eye. Topography showed marked flattening of the left cornea, and simulated retinal images of Landolt C circles were distorted in the left eye. CONCLUSIONS: This is a rare case in which radial keratotomy induced severe hyperopic shift. Wavefront analysis with a Hartmann-Shack aberrometer revealed severe irregular astigmatism with small clear zones.

Subconjunctival hemorrhage and conjunctivochalasis.

OBJECTIVE: Subconjunctival hemorrhage (SCH) is a relatively common disease, but there have been no reports concerning the relationship between SCH and conjunctivochalasis (CCh). We compared the grade of CCh between patients with SCH and control patients. DESIGN: Prospective, nonrandomized study. PARTICIPANTS: A total of 104 patients with SCH aged 41 to 94 years and 120 age- and gender-matched controls aged 41 to 94 years were enrolled. METHODS: The conjunctiva was divided into the following 8 equal areas: superior, superior/nasal, nasal, inferior/nasal, inferior, inferior/temporal, temporal, and superior/temporal. The age, gender, medical history, ocular history, site of hemorrhage, grade of CCh at 3 locations (nasal, middle, and temporal), and other parameters of CCh were determined in all subjects. MAIN OUTCOME MEASURES: Grade of each CCh parameter and location of SCH. RESULTS: The mean grade of CCh was higher in patients with SCH than in control patients at the nasal (P<0.00001), middle (P<0.00001), and temporal areas (P<0.00001). The downward gaze- or digital pressure-dependent changes of CCh and the frequency of superficial punctate keratitis were all increased in SCH patients compared with control patients (P<0.00001, P<0.00001, and P = 0.00106, respectively). The number of areas involved by SCH and the presence of SCH in each area were positively correlated with the grade of each CCh-related parameter (P<0.05). CONCLUSIONS: This was the first assessment of the grade of CCh in a large series of consecutive patients with SCH. Our results strongly suggest that CCh may have an important role in the pathogenesis of SCH.

Conjunctivochalasis and contact lenses.

PURPOSE: To assess the relationship between age and the incidence and severity of conjunctivochalasis in contact lens (CL) wearers by grading of conjunctivochalasis, and to compare the severity of conjunctivochalasis between CL wearers and nonwearers. DESIGN: Prospective, nonrandomized consecutive case study. METHODS: A total of 600 CL wearers (94 hard CLs [HCL] and 506 soft CLs [SCL]) aged 11 to 60 years and 579 nonwearers aged 10 to 60 years were enrolled. The age, gender, medical history, ocular history, and the grade and other parameters of conjunctivochalasis at 3 locations (nasal, middle, and temporal areas) were determined in all subjects. RESULTS: The prevalence of conjunctivochalasis increased dramatically with age in all groups. The mean grade of conjunctivochalasis was higher in CL wearers than in nonwearers (nasal area, P < .00001; temporal area, P < .00001) and was higher in HCL wearers than in SCL wearers (nasal area, P < .00001; temporal area, P = .00003). Parameters such as the downward gaze-dependent or digital pressure-dependent changes of conjunctivochalasis and the presence of superficial punctate keratitis all increased with age in both CL wearers and nonwearers. The gaze-dependent and pressure-dependent changes of conjunctivochalasis showed an increase with age and the duration of CL wear in both SCL and HCL wearers. CONCLUSIONS: This was the first assessment of the severity of conjunctivochalasis in a large series of consecutive CL wearers. Our results strongly suggest that wearing CLs is an important risk factor for conjunctivochalasis.

Changes of conjunctivochalasis with age in a hospital-based study.

PURPOSE: To assess the relationship between age and the incidence and severity of conjunctivochalasis by using the grading system for conjunctivochalasis proposed by Meller and Tseng. DESIGN: Prospective, nonrandomized, consecutive case study. METHODS: A total of 1,416 patients aged one to 94 years were enrolled. The age, gender, medical history, ocular history, and grade and parameters of conjunctivochalasis at three locations (nasal, middle, and temporal) were determined in all subject. RESULTS: The prevalence of conjunctivochalasis increased dramatically with age (one to 10 years, 6.8%; 11 to 20 years, 36.2%; 21 to 30 years, 61.5%; 31 to 40 years, 71.4%; 41 to 50 years, 90.2%; 51 to 60 years, 94.2%; 61 to 70 years, 98.0%; 71 to 80 years, 99.0%; 81 to 90 years, 98.5%; and 91 to 100 years, 100.0%). The mean grade of conjunctivochalasis was higher in female patients than in male patients (nasal area, P = .0229; temporal area, P = .0078) and was higher for the temporal conjunctiva than the nasal conjunctiva (P < .0000001). Parameters such as the downward gaze- or digital pressure-dependent changes of conjunctivochalasis and the presence of superficial punctate keratitis all increased with age. These conjunctivochalasis-related parameters were correlated with the severity of conjunctivochalasis (P < .00001), especially of the middle bulbar conjunctiva. CONCLUSIONS: We report the grade of conjunctivochalasis in a large series of consecutive patients. Our results strongly suggest that conjunctivochalasis is an age-dependent eye disease that occurs in young adulthood and thereafter. The present grading system should be useful for future clinical studies of conjunctivochalasis.

Nontraumatic keratomycosis caused by Alternaria in a glaucoma patient.

A case of keratomycosis caused by Alternaria species in a patient with glaucoma is reported. A 55-year-old Japanese man who had been followed for developmental glaucoma presented with corneal ulcer in his right eye, which did not respond to antibacterial agents. There was no history of traumatic episode. Culture on potato dextrose agar from corneal scraping yielded Alternaria species. Topical amphotericin B treatment achieved recovery from ulceration although the corneal opacity remained.

In vivo confocal microscopy of hereditary sensory and autonomic neuropathy.

PURPOSE: To observe the morphology of the corneal cells and corneal nerve fibers in patients with type IV or V hereditary sensory and autonomic neuropathy (HSAN) by in vivo confocal microscopy and elucidate the mechanism leading to the loss of corneal sensation in this disease. METHODS: In vivo confocal microscopy was performed on the central cornea of the right eye in 3 patients with HSAN (ages 17, 20, and 32 years), and their corneal morphology was compared with that of 3 healthy subjects (ages 28, 30, and 36 years). Corneal sensation was tested with a Cochet-Bonnet esthesiometer. RESULTS: The superficial epithelial cell density was lower in the HSAN patients compared with the healthy subjects (1525, 1225, and 1250/mm(2) vs. 2225, 1750, and 2500/mm(2)), but the basal epithelial cell density of the patients was similar to that of the healthy subjects. Nerve bundles were clearly observed in the sub-basal nerve plexus layer of the cornea in the healthy subjects, but were undetectable at the central cornea in the patients with HSAN. The corneal sensation of the patients with HSAN was much weaker than that of the healthy subjects (2.79, 40.30, and 132.50 g/mm(2) vs. 1.47, 1.47, and 1.47 g/mm(2)). CONCLUSIONS: Superficial keratopathy accompanied with neurotrophic keratopathy and tear film instability observed clinically agrees with the large keratinized cells in the superficial corneal epithelium by in vivo confocal microscopy in these patients. Our findings suggest that the loss of corneal nerves contributes to impairment of corneal sensation in patients with type IV or V HSAN.

In vivo confocal microscopy of human cornea covered with human amniotic membrane.

PURPOSE: Amniotic membrane transplantation has been widely performed to reconstruct the surface of the eye and treat chemical burns or epithelial defects. However, we have difficulty observing the cornea through the opaque transplanted amniotic membrane by slit-lamp biomicroscopy. We investigated the use of confocal microscopy for observation of human corneas covered with amniotic membrane. METHODS: Human amniotic membrane was placed onto the normal corneas of five volunteers aged 22-24 years. Then, all layers of the covered corneas were observed by in vivo confocal microscopy. RESULTS: Confocal microscopy displayed the epithelium, basement membrane, and stroma of the amniotic membrane. It also displayed the corneal epithelium. Furthermore, corneal stromal keratocytes and the corneal endothelium were clearly observed through the amniotic membrane by confocal microscopy. CONCLUSIONS: We demonstrated that in vivo confocal microscopy enabled us to observe all layers of corneas covered with amniotic membrane in normal human eyes. Our findings suggest that confocal microscopy may have advantages for clinical examination of the ocular surface, including all layers of the cornea.

Transplantation of a sheet of human corneal endothelial cell in a rabbit model.

PURPOSE: To develop a novel method for constructing a sheet of human corneal endothelial cells (HCECs) and examine the properties of the HCEC sheet. METHODS: HCECs were cultured on a cell culture insert for a week; ethylenediamine tetraacetic acid was applied from the bottom of the cell culture insert to attenuate the attachment of HCECs. The sheet of HCECs was constructed by bluntly detaching the cell sheet with a spatula. HCEC cell sheets were placed on the posterior surface of excised rabbit corneal buttons and transplanted onto the corneal beds of donor rabbits. In two eyes from the HCEC sheet group, cultured HCECs were labeled with PKH26 to observe the localization of HCECs after transplantation. RESULTS: Cultured HCECs could be bluntly detached en bloc from the bottom of a culture insert. Immunostaining for ZO-1, Na+, K+-ATPase, laminin, fibronectin, and type IV collagen was positive in the cell sheet. The average cell density in a HCEC sheet was 2,425 cells/mm(2). After HCEC sheet transplantation, corneal edema decreased much earlier in the HCEC group than in the control group. In the HCEC sheet group, the monolayer of continuous cells attached to the posterior surface of the transplanted rabbit cornea and the posterior surface of transplanted cornea was covered with PKH26-labeled cells. The average endothelial cell density in the HCEC sheet group seven days postoperatively was 2,244 cells/mm(2). CONCLUSIONS: This technique for producing an HCEC sheet might be useful in regenerative medicine for the cornea and reconstruction of the corneal endothelium.