Rabbit Fungal Keratitis Model of Fusarium solani Tested Against Three Commercially Available Antifungal Drugs.

OBJECTIVES: The purpose of this study was to develop a reproducible preclinical Fusarium solani keratitis model, which would allow comparative testing of currently available antifungals (NATACYN [Alcon, Fort Worth, TX], voriconazole 1%, and amphotericin B 0.1%) as well as efficacy testing of new antifungals for translation into clinical practice in the future. METHODS: The rabbit F. solani keratitis model was developed in New Zealand white rabbits using local and systemic immunosuppression. Infection was introduced by intrastromal injection of F. solani spores into one of the immunosuppressed rabbit eyes while the contralateral eye was a control. Progress of the infection was assessed by the clinical features, histopathology, and viable fungal counts. In this study, the efficacy of currently available antifungals (NATACYN [Alcon], voriconazole 1%, and amphotericin B 0.1%) was compared. Rabbits were randomly divided (n=4 in each group), and the respective antifungal was instilled topically 5 times/day for 7 days. Treatment effects were analyzed by evaluating the anterior segment with the help of slit-lamp, histopathological findings and viable fungal culture at the end of the experiment. RESULTS: We report the development of a reproducible and progressive rabbit F. solani keratitis model as shown by the substantial viable fungal counts (3 log CFU), the presence of large patchy lesions and substantial hypopyon in the 12-day model correlated with specific histopathological analysis for fungus (extended F. solani hyphae from midcorneal stroma into the anterior chamber and traverse Descemet membrane with anterior chamber suppurative plaque). Voriconazole 1% and NATACYN revealed significant reduction of the fungal wound area (P=0.02 and 0.021), respectively, while amphotericin B 0.1% exhibited P value of 0.083 compared with their infected nontreated controls. Voriconazole 1% and amphotericin B 0.1% showed significant viable fungal count differences (P=0.004 and 0.01), respectively, whereas P value of NATACYN was 0.337 compared with control infected corneas. CONCLUSION: The reported rabbit fungal keratitis model can be used for screening new antifungals and evaluating currently available antifungals to facilitate better clinical outcomes. Voriconazole 1% showed the best efficacy among the three tested currently available antifungals by showing the significant differences in both wound size and viable fungal count comparisons in our F. solani rabbit keratitis model.

Mutations in sodium-borate cotransporter SLC4A11 cause recessive congenital hereditary endothelial dystrophy (CHED2).

Congenital hereditary endothelial dystrophy (CHED) is a heritable, bilateral corneal dystrophy characterized by corneal opacification and nystagmus. We describe seven different mutations in the SLC4A11 gene in ten families with autosomal recessive CHED. Mutations in SLC4A11, which encodes a membrane-bound sodium-borate cotransporter, cause loss of function of the protein either by blocking its membrane targeting or nonsense-mediated decay.

Rapid bactericidal action of alpha-mangostin against MRSA as an outcome of membrane targeting.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has created the need for better therapeutic options. In this study, five natural xanthones were extracted and purified from the fruit hull of Garcinia mangostana and their antimicrobial properties were investigated. α-Mangostin was identified as the most potent among them against Gram-positive pathogens (MIC=0.78-1.56 µg/mL) which included two MRSA isolates. α-Mangostin also exhibited rapid in vitro bactericidal activity (3-log reduction within 5 min). In a multistep (20 passage) resistance selection study using a MRSA isolated from the eye, no resistance against α-mangostin in the strains tested was observed. Biophysical studies using fluorescence probes for membrane potential and permeability, calcein encapsulated large unilamellar vesicles and scanning electron microscopy showed that α-mangostin rapidly disrupted the integrity of the cytoplasmic membrane leading to loss of intracellular components in a concentration-dependent manner. Molecular dynamic simulations revealed that isoprenyl groups were important to reduce the free energy for the burial of the hydrophobic phenyl ring of α-mangostin into the lipid bilayer of the membrane resulting in membrane breakdown and increased permeability. Thus, we suggest that direct interactions of α-mangostin with the bacterial membrane are responsible for the rapid concentration-dependent membrane disruption and bactericidal action.

Nerve growth factor expression and nerve regeneration in monkey corneas after LASIK.

PURPOSE: To determine whether changes in cornea nerve growth factor (NGF) protein and gene expression correlate with corneal nerve regeneration after LASIK in rhesus monkeys. METHODS: Ten rhesus monkeys underwent randomly assigned LASIK procedures to correct -8.0 diopters of myopia in both eyes and two additional monkeys (with no surgery) served as controls. The central corneas of the experimental animals were excised 3 and 7 days and 1, 3, and 6 months after the surgery. Nerve regeneration, NGF mRNA, and protein expression were analyzed by gold chloride staining, real-time polymerase chain reaction, and enzyme-linked immune sorbent assay analysis, respectively. RESULTS: Compared to controls, the LASIK-treated animals had a significantly diminished nerve plexus in the sub-basal region of the cornea at all times after the surgery (P < .001). NGF protein levels decreased significantly on days 3 and 7 after LASIK (P < .001), but returned to control levels 1 month later. NGF mRNA levels increased 5.4-fold on day 3 after the surgery (P < .001), then reduced to two-fold (P < .05) above control levels on day 7 and were back to normal at 3 months and beyond. After LASIK, the changes of early NGF protein and NGF mRNA levels correlated with the density of the corneal nerve plexuses. CONCLUSIONS: The results showed that the LASIK procedure in non-human primates was associated with changes in NGF protein and mRNA levels in the cornea. Such changes may be related to the initiation of nerve regeneration and the final recovery of nerve plexuses in the cornea.

Definition and Diagnostic Criteria for Pediatric Blepharokeratoconjunctivitis.

Importance: Pediatric blepharokeratoconjunctivitis (PBKC) is a chronic, sight-threatening inflammatory ocular surface disease. Due to the lack of unified terminology and diagnostic criteria, nonspecific symptoms and signs, and the challenge of differentiation from similar ocular surface disorders, PBKC may be frequently unrecognized or diagnosed late. Objective: To establish a consensus on the nomenclature, definition, and diagnostic criteria of PBKC. Design, Setting, and Participants: This quality improvement study used expert panel and agreement applying the non-RAND modified Delphi method and open discussions to identify unified nomenclature, definition, and definitive diagnostic criteria for PBKC. The study was conducted between September 1, 2021, and August 14, 2022. Consensus activities were carried out through electronic surveys via email and online virtual meetings. Results: Of 16 expert international panelists (pediatric ophthalmologists or cornea and external diseases specialists) chosen by specific inclusion criteria, including their contribution to scientific leadership and research in PBKC, 14 (87.5%) participated in the consensus. The name proposed was "pediatric blepharokeratoconjunctivitis," and the agreed-on definition was "Pediatric blepharokeratoconjunctivitis is a frequently underdiagnosed, sight-threatening, chronic, and recurrent inflammatory eyelid margin disease associated with ocular surface involvement affecting children and adolescents. Its clinical spectrum includes chronic blepharitis, meibomitis, conjunctivitis, and corneal involvement ranging from superficial punctate keratitis to corneal infiltrates with vascularization and scarring." The diagnostic criteria included 1 or more suggestive symptoms accompanied by clinical signs from 3 anatomical regions: the eyelid margin, conjunctiva, and cornea. For PBKC suspect, the same criteria were included except for corneal involvement. Conclusions and Relevance: The agreements on the name, definition, and proposed diagnostic criteria of PBKC may help ophthalmologists avoid diagnostic confusion and recognize the disease early to establish adequate therapy and avoid sight-threatening complications. The diagnostic criteria rely on published evidence, analysis of simulated clinical cases, and the expert panel's clinical experience, requiring further validation with real patient data analysis.

Corneal transplantation.

Corneal transplantation or keratoplasty has developed rapidly in the past 10 years. Penetrating keratoplasty, a procedure consisting of full-thickness replacement of the cornea, has been the dominant procedure for more than half a century, and successfully caters to most causes of corneal blindness. The adoption by specialist surgeons of newer forms of lamellar transplantation surgery, which selectively replace only diseased layers of the cornea, has been a fundamental change in recent years. Deep anterior lamellar keratoplasty is replacing penetrating keratoplasty for disorders affecting the corneal stromal layers, while eliminating the risk of endothelial rejection. Endothelial keratoplasty, which selectively replaces the corneal endothelium in patients with endothelial disease, has resulted in more rapid and predictable visual outcomes. Other emerging therapies are ocular surface reconstruction and artificial cornea (keratoprosthesis) surgery, which have become more widely available because of rapid advances in these techniques. Collectively, these advances have resulted in improved outcomes, and have expanded the number of cases of corneal blindness, which can now be treated successfully. Femtosecond-laser-assisted surgery, bioengineered corneas, and medical treatment for endothelial disease are also likely to play a part in the future.

A mouse model of corneal endothelial decompensation using cryoinjury.

PURPOSE: To develop a mouse model of bullous keratoplasty and evaluate the safety and efficacy of cryoinjury-induced corneal endothelial decompensation. METHODS: Transcorneal freezing was performed on the right eye of each mouse. One cycle of cryoinjury was performed in 18 eyes (group A), and three cycles were performed in 17 eyes (group B). Pachymetry and intraocular pressure (IOP) measurements were done preoperatively, as well as at 1, 3, 7, 14, and 21 days after cryoinjury. At each post-cryoinjury time point, three mice from each group were euthanized, and the corneas underwent histology and electron microscopy. RESULTS: In both groups, significant corneal edema was noted at post-cryoinjury day 1, which was maintained throughout the study period. IOP remained within normal range in group A, but increased significantly with time in group B (p=0.011 at day 1, 0.038 at day 3, 0.026 at day 14, and 0.008 at day 21). In group B, serious complications including hyphema (one case), severe iridocorneal adhesion (15 cases), and total cataract (three cases) were detected, while only mild iridocorneal adhesion (four cases) and cataract (three cases) were noted in group A. Live/dead cell assay, hematoxylin and eosin staining, and scanning electron microscopy revealed successful ablation of corneal endothelial cells and absence of regeneration in both groups. Hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated nick end labeling assay showed that apoptosis was mainly confined to the posterior stroma and endothelium in group A, while severe apoptosis was observed throughout all layers of the cornea in group B. CONCLUSIONS: One cycle of cryoinjury was safer than three, while both were equally effective in inducing bullous keratopathy. This cryoinjury mouse model of bullous keratopathy was a consistently reproducible model that can be used for further studies on endothelial cell damage and rescue therapy.

Cost-effectiveness of Descemet's stripping endothelial keratoplasty versus penetrating keratoplasty.

PURPOSE: Selective endothelial transplantation in the form of Descemet's stripping endothelial keratoplasty (DSEK) is rapidly replacing traditional full-thickness penetrating keratoplasty (PK) for endothelial disease. An incremental cost-effectiveness analysis was performed to determine whether the benefits of DSEK are worth the additional costs. DESIGN: Retrospective cohort study. PARTICIPANTS: Patients at the Singapore National Eye Center, a tertiary eye center in Singapore, with Fuchs' dystrophy or bullous keratopathy who underwent either PK or DSEK. INTERVENTION: Patients underwent either PK (n = 171) or DSEK (n = 93) from January 2001 through December 2007. Data were collected from inpatient and outpatient notes corresponding to the time immediately before the procedure to up to 3 years after. MAIN OUTCOME MEASURES: Improvements in best spectacle-corrected visual acuity were used to calculate the increase in quality-adjusted life years (QALYs) 3 years after the procedure. This was combined with hospital charges (a proxy for costs) to determine incremental cost-effectiveness ratios (ICERs) comparing PK with no intervention and DSEK with PK. RESULTS: Three-year charges for DSEK and PK were $7476 and $7236, respectively. The regression-adjusted improvement in visual acuity for PK relative to no intervention was -0.613 logarithm of the minimum angle of resolution (logMAR) units (P<0.001), and for DSEK relative to PK, it was -0.199 logMAR units (P = 0.045). The regression-adjusted marginal gain in utility for PK relative to no intervention was 0.128 QALYs (P<0.001) and for DSEK relative to PK was 0.046 QALYs (P = 0.031). This resulted in ICERs of $56 409 per QALY for PK relative to no intervention and $5209 per QALY for the more expensive DSEK relative to PK. CONCLUSIONS: If the goal is to maximize societal health gains given fixed resources, DSEK should be the preferred strategy. For a fixed budget, it is possible to achieve greater QALY gains by providing DSEK to as many patients as possible (and nothing to others), rather than providing PK.

Comparison of four different VisuMax circle patterns for flap creation after small incision lenticule extraction.

PURPOSE: To compare four different Circle patterns for flap creation after small incision lenticule extraction (SMILE). METHODS: SMILE was performed on six rabbits. Twenty-eight days after the initial procedure, corneal flaps were created using Circle patterns. Rabbits were divided into four groups (patterns A, B, C, and D). Pattern A creates a circular side cut to meet the cap cut within the clearance zone (outside of the optical zone). Patterns B, C, and D create a lamellar ring posterior, anterior, and at the same depth, respectively, to the cap to meet the cap cut in the clearance zone with the help of a junction cut. Difficulty of flap lift was graded from 1 (easiest) to 5 (most difficult). The bed quality was assessed by scanning electron microscopy. RESULTS: Flaps created by patterns A and D were the easiest to lift (grade 2). The resulting flap bed was smooth and undisrupted. However, pattern A resulted in a reduced re-treatment area. Flaps created by pattern B were the most difficult to lift (grade 4). The stromal dissection was difficult in an attempt to ascertain the original optical zone from the lamellar ring, placed posterior. Flaps produced by pattern C were easy to lift, with minor intrastromal resistance experienced during the lifting process (grade 3). The transition between the lamellar ring and cap cut was hardly discernible in pattern C-treated corneas. CONCLUSIONS: Pattern D, a lamellar ring adjacent to the cap cut, was the most optimal to be used for flap creation in cases of SMILE re-treatment.

In vivo biocompatibility of two PEG/PAA interpenetrating polymer networks as corneal inlays following deep stromal pocket implantation.

This study compared the effects of implanting two interpenetrating polymer networks (IPNs) into rabbit corneas. The first (Implant 1) was based on PEG-diacrylate, the second (Implant 2) was based on PEG-diacrylamide. There were inserted into deep stromal pockets created using a manual surgical technique for either 3 or 6 months. The implanted corneas were compared with normal and sham-operated corneas through slit lamp observation, anterior segment optical coherence tomography, in vivo confocal scanning and histological examination. Corneas with Implant 1 (based on PEG-diacrylate) developed diffuse haze, ulcers and opacities within 3 months, while corneas with Implant 2 (based on PEG-diacrylamide) remained clear at 6 months. They also exhibited normal numbers of epithelial cell layers, without any immune cell infiltration, inflammation, oedema or neovascularisation at post-operative 6 month. Morphological studies showed transient epithelial layer thinning over the hydrogel inserted area and elevated keratocyte activity at 3 months; however, the epithelium thickness and keratocyte morphology were improved at 6 months. Implant 2 exhibited superior in vivo biocompatibility and higher optical clarity than Implant 1. PEG-diacrylamide-based IPN hydrogel is therefore a potential candidate for corneal inlays to correct refractive error.

Indications, outcomes, and risk factors for failure in tectonic keratoplasty.

PURPOSE: Outcomes of corneal transplantation for tectonic indications and risk factors for (tectonic and physiologic) graft failure. DESIGN: Retrospective cohort study. PARTICIPANTS: Consecutive patients who underwent keratoplasty for tectonic indications at the Singapore National Eye Centre (SNEC) between January 1, 1991, and December 1, 2009. METHODS: Clinical data and donor and recipient characteristics were recorded and analyzed from subjects in the prospective Singapore Corneal Transplant Study. MAIN OUTCOME MEASURES: (1) Tectonic (anatomic) failure defined as recurrence of corneal melt threatening tectonic integrity and requiring additional corneal grafting within 3 months of the primary procedure. (2) Physiologic failure defined as irreversible change in graft clarity preventing recovery in useful vision in grafts initially clear 2 weeks postoperatively. RESULTS: The mean age of the study cohort (n = 362, 193 male and 169 female subjects) was 51.5 ± 20.2 years, with a mean follow-up of 25.8 ± 18.7 months. Patients underwent penetrating keratoplasty (PK) (n = 142, 39.2%), anterior lamellar keratoplasty (ALK) (n = 127, 35.1%), or a peripheral corneoscleral patch graft (n = 93, 25.7%) most commonly for inflammation (n = 68, 18.8%), trauma (n = 66, 18.2%), or infection (n = 66, 18.2%). Risk factors for tectonic failure (18/362 eyes, 5.0%) were severe lid disease (odds ratio [OR], 6.1; 95% confidence interval [CI], 1.7-22.1; P = 0.006), central ALK (OR, 7.5; 95% CI, 1.8-32.4; P = 0.007), and peripheral grafts (OR, 5.7; 95% CI, 1.1-28.3; P = 0.035). Among anatomically successful central grafts (n = 223), the mean physiological graft survival was 96 months (95% CI, 83-110); Kaplan-Meier probabilities for survival at 10 years were 66.8% for ALK and 44.2% for PK. Active corneal inflammation (hazard ratio [HR], 2.5; 95% CI, 1.4-4.4; P = 0.003) and larger donor and recipient graft sizes of ≥ 9 mm (HR, 17.9; 95% CI, 2.3-140.3; P = 0.006) were risk factors for physiologic graft failure in anatomically successful eyes with central tectonic grafts. CONCLUSIONS: Patients with lid disease, central ALK, and peripheral grafts were at higher risk of anatomic failure. For anatomically successful cases with central tectonic grafts, active corneal inflammation and donor size ≥ 9 mm were risk factors for physiologic failure. In these cases, our results suggest that ALK had better physiologic graft survival outcomes than PK.

Prevalence of and racial differences in pterygium: a multiethnic population study in Asians.

PURPOSE: To describe the prevalence and risk factors of pterygium in a multiethnic Asian population and to examine racial differences. DESIGN: Population-based study in Singapore, located 1° north of the equator. PARTICIPANTS: Data were analyzed from 8906 participants from 3 population-based studies of Malays, Indians, and Chinese persons 40 years of age and older conducted between 2004 and 2011. METHODS: Standardized slit-lamp examinations were performed by trained study ophthalmologists to examine the anterior segment for evidence of pterygium. Every subject underwent standardized systemic and ocular examinations, interviewer-administered questionnaires, and blood investigations for risk factor assessment. Regression and principle component analysis models were constructed to study the relationship of race and other factors to pterygium. MAIN OUTCOME MEASURES: Any pterygium and severe (grade 3 or opaque) pterygium. RESULTS: The overall prevalence of any pterygium was 10.1% (n = 900), of which severe pterygium was seen in 1.6% (n = 142). The prevalence of any pterygium was more common in Malays (15.5%) than Chinese (7.0%; P<0.001) or Indians (7.0%; P<0.001). Multivariate analysis revealed increasing age (P<0.001), male gender (P<0.001), Malay race (P<0.001), and having a poorer education level (P<0.001) as significant factors for any pterygium. Race contributed significantly to presence of any pterygium (41%; P<0.001) or presence in both eyes (33%; P<0.001) compared with other risk factors. Severe pterygium was associated with outdoor occupation (P = 0.02), but race was not a significant risk factor in multivariate analysis. CONCLUSIONS: This population-based study in Asian persons of different races living in the same geographical location at the equator indicated that race is a significant risk factor for pterygium, with Malays having higher prevalence than Indians and Chinese, while controlling for other risk factors.

Correlation of anterior segment optical coherence tomography measurements with graft trephine diameter following descemet stripping automated endothelial keratoplasty.

BACKGROUND: To assess repeatability of the Zhongshan Assessment Program (ZAP) software measurement of Anterior Segment Optical Coherence Tomography (ASOCT) images and correlate with graft trephine diameter following Descemet Stripping Automated Endothelial Keratoplasty (DSAEK) METHODS: Retrospectively evaluated interventional case series. 121 consecutive eyes undergoing DSAEK over a 26 month period underwent ASOCT imaging 1 month after their surgery. ASOCT images were processed using ZAP software which measured the graft and cornea parameters including anterior and posterior graft arc length and cord length, posterior cornea arc length (PCAL) and anterior chamber width. RESULTS: The graft measurements showed good repeatability on ASOCT using ZAP with high intra class coefficient and small variation in the coefficient of variation. On ASOCT, the mean recipient PCAL was 12.99+/-0.69 mm and the anterior chamber width was 11.16+/-0.57 mm. The mean Graft anterior arc length was 9.69+/-0.66 mm and the mean Graft anterior cord length was 8.92+/-2.94 mm. The mean graft posterior arc length was 9.24+/-0.75 mm and the mean graft posterior cord length was 8.15+/-0.57 mm. Graft posterior arc length (rho=0.788, p< 0.001) correlated best with intra-operative graft trephine diameter. The mean ratio of posterior graft arc length to PCAL was 0.712 +/- 0.056. CONCLUSIONS: We have validated the repeatability of the ZAP software for DSAEK graft measurements from ASOCT images and shown that the graft arc length parameters calculated from the ASOCT images correlate well with the intra-operative graft trephine diameter. This software may help surgeons determine the optimal DSAEK graft size based on pre-operative ASOCT measurements of the recipient eye.

Reproducibility of Corneal Graft Thickness measurements with COLGATE in patients who have undergone DSAEK (Descemet Stripping Automated Endothelial Keratoplasty).

BACKGROUND: The CorneaL GrAft Thickness Evaluation (COLGATE) system was recently developed to facilitate the evaluation of corneal graft thickness from OCT images. Graft thickness measurement can be a surrogate indicator for detecting graft failure or success. The purpose of this study was to determine the reproducibility of the COLGATE system in measuring DSAEK graft area between two observers. METHODS: This was a prospective case series in which 50 anterior segment OCT images of patients who had undergone DSAEK in either eye were analysed. Two observers (MW, AC) independently obtained the image analysis for the graft area using both semi automated and automated method. One week later, each observer repeated the analysis for the same set of images. Bland-Altman analysis was performed to analyze inter and intra observer agreement. RESULTS: There was strong intraobserver correlation between the 2 semi automated readings obtained by both observers. (r = 0.936 and r = 0.962). Intraobserver ICC for observer 1 was 0.936 (95% CI 0.890 to 0.963) and 0.967 (95% CI 0.942 to 0.981) for observer 2. Likewise, there was also strong interobserver correlation (r = 0.913 and r = 0.969). The interobserver ICC for the first measurements was 0.911 (95% CI 0.849 to 0.949) and 0.968 (95% CI 0.945 to 0.982) for the second. There was statistical difference between the automatic and the semi automated readings for both observers (p = 0.006, p = 0.003). The automatic readings gave consistently higher values than the semi automated readings especially in thin grafts. CONCLUSION: The analysis from the COLGATE programme can be reproducible between different observers. Care must be taken when interpreting the automated analysis as they tend to over estimate measurements.

Osteo-odonto keratoprosthesis: systematic review of surgical outcomes and complication rates.

Case series on osteo-odonto keratoprosthesis (OOKP) published in English from 1950-June 2010 were identified in Medline/PubMed. Indications for surgery, visual acuity, anatomical survival, complication and repeat surgery rates were compared among the different studies. Our own case series is a retrospective review of all OOKP surgeries performed in our center from February 2004-July 2011. Eight case series including our own were systematically reviewed. Sample sizes ranged from 4-181 eyes. The most common indications for surgery were severe cases of Stevens-Johnson syndrome and thermal and chemical burns that were unamenable to other forms of surgery or had had previous surgical failure. Anatomical survival rate in all the studies was 87.8% (range 67-100%) at 5 years, and three studies showed survival rates of 81.0% (range 65-98%) at 20 years. Visual acuity was more than 6/18 in 52% (range 46-72%) of the eyes with OOKP surgery. The most common intraoperative complication was vitreous hemorrhage (0-52%) and the most common long-term blinding complication was glaucoma (7-47%). Endophthalmitis rates ranged from 2-8%. The most common repeat surgical procedure was mucosal trimming due to mucosal overgrowth at the optical cylinder and mucosal grafting for extrusion of the OOKP or mucosal ulceration. Of the available biological and synthetic keratoprosthesis, OOKP appears to be an excellent option for the treatment of end-stage corneal diseases.

S100A expression in normal corneal-limbal epithelial cells and ocular surface squamous cell carcinoma tissue.

PURPOSE: To study the expression and cellular distribution of multiple S100A genes and proteins in normal corneal-limbal epithelium and ocular surface squamous cell carcinoma (SCC) tissue. METHODS: Normal corneal-limbal tissue was obtained from the Lions Eye Bank, Tampa, FL. Ocular surface SCC tissues were excised from patients undergoing surgery at Singapore National Eye Centre. S100A mRNA expression was measured by quantitative PCR. S100 protein distribution was determined by immunofluorescent staining analysis. RESULTS: Twelve S100 mRNAs were identified in human corneal and limbal epithelial cells. S100A2, A6, A8, A9, A10, and A11 mRNA was expressed at high level, while S100A1, A3, A4, A5, A6, A7, and A12 mRNA expression was low. The intracellular localization of S100A2, A6, A8, A9, A10 and A11 protein was determined in normal corneal-limbal and SCC tissues. S100A2 and S100A10 proteins were enriched in basal limbal epithelial cells of the normal tissue. S100A8 and S100A9 were found only at the surface of peripheral corneal and limbal epithelium. S100A6 was uniformly found at the plasma membrane of corneal and limbal epithelial cells. S100A11 was found at the supralayer limbal epithelial cells adjacent to the conjunctiva. SCC tissue showed typical pathological changes with expression of cytokeartin (CK) 14 and CK4 in the epithelial cells. All SCC epithelial cells were positive of S100A2, S100A10, S100A6 and S100A11 staining. Intracellular staining of S100A8 and S100A9 was found in several layers of SCC epithelium. Expression of S100A2 and S100A10 decreased dramatically in cultured limbal epithelial cells with increased passaging, which was accompanied by a small increase of S100A9 mRNA, with no changes of S100A8 gene expression. Serum and growth hormone depletion of the culture serum caused a small reduction of S100A2 and S100A10 gene expression, which was accompanied by a small increase of S100A9 mRNA while no changes of S100A8 expression was measured. CONCLUSIONS: Normal corneal and limbal epithelial cells express a broad spectrum of S100 genes and proteins. Ocular surface SCC express high levels of S100A2, S100A10, S100A8 and S100A9 proteins. The expression of S100A2 and S100A10 is associated with limbal epithelial cell proliferation and differentiation.

Cornea lenticule viability and structural integrity after refractive lenticule extraction (ReLEx) and cryopreservation.

PURPOSE: To assess and compare keratocyte viability and collagen structure in cornea stroma lenticules collected immediately after refractive lenticule extraction (ReLEx) and one month after cryopreservation. METHODS: The fresh and cryopreserved human stroma lenticules procured after ReLEx were processed for ultrastructural analysis of keratocytes and collagen fibrils with transmission electron microscopy (TEM), apoptotic cell detection with deoxynucleotidyl transferase-mediated nick end labeling assay (TUNEL) assay, and cultured for keratocyte-specific gene expression analysis using reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: The periphery of the lenticule had greater TUNEL-positive cells compared to the center of the lenticule in both fresh and cryopreserved groups. There was an increase in TUNEL-positive cells after cryopreservation, which was significantly higher in the center of the lenticule, but not in the periphery. TEM showed apoptotic, necrotic and viable quiescent keratocytes in fresh and cryopreserved lenticules. Collagen analysis with TEM showed a well preserved and well aligned structure in fresh and cryopreserved lenticules; without significant change in the total number of collagen fibrils but with an increased collagen fibril density (CFD) after cryopreservation. In vitro, isolated keratocytes derived from fresh and cryopreserved lenticules exhibited a typical fibroblastic phenotype. RT-PCR showed a positive gene expression for keratocan (KERA) and aldehyde dehydrogenase 3A1 (ALDH3A1) in cells isolated from fresh and cryopreserved lenticules. CONCLUSIONS: The stromal lenticules extracted from ReLEx surgery remain viable after cryopreservation. Although they showed a decrease in CFD, the collagen architecture was preserved and there was good cellular viability.

Incidence and characteristics of unilateral keratoconus classified on corneal topography.

PURPOSE: To evaluate the characteristics of unilateral keratoconus defined on the basis of corneal topography and analyze videokeratography parameters between fellow eyes and normal controls. METHODS: A total of 111 patients with clinical keratoconus were prospectively enrolled. Both eyes were evaluated with Tomey (Tomey Corp) and Orbscan II (Bausch & Lomb) corneal topography systems. The patient was classified as having unilateral keratoconus if one eye had clinical keratoconus and the other eye did not have any topographic signs of keratoconus such as asymmetric videokeratographic pattern, positive result in Tomey keratoconus screening, maximum posterior elevation >40 µm, or corneal thinnest pachymetry <500 µm. Clinical characteristics and 13 Orbscan II quantitative indices between keratoconic and fellow eyes and normal control eyes were evaluated. RESULTS: Five (4.5%) of 111 patients with keratoconus had no topographic evidence of keratoconus in the fellow eye. All clinically normal fellow eyes had symmetric bowtie patterns. Statistically significant differences were noted in maximum posterior elevation, corneal irregularity, and corneal thinnest values between keratoconic eyes and fellow eyes, and between keratoconic eyes and control eyes. Only 3-mm irregularity was significantly higher in the fellow eyes compared with control eyes (P<.05). An increased trend for corneal 5-mm irregularity was found in fellow eyes compared to control eyes. CONCLUSIONS: The incidence of unilateral keratoconus was 4.5%. A trend of higher irregularity was found in fellow eyes compared with control eyes. This finding indicates that fellow eyes may show a certain low-expressivity morphologic feature of keratoconus.

Evolution of deep anterior lamellar keratoplasty (DALK).

The concept of selective tissue transplantation was theorized over two centuries ago. However, deep anterior lamellar keratoplasty (DALK) was difficult to perform with the instruments available at that time, and visual acuity results were poor compared to those achieved by full-thickness transplantation. Recently, many technical improvements in surgical instrumentation and advances in eye bank technology have led to the re-emergence of DALK. Now, patients with stromal pathologies not affecting the corneal endothelium can benefit from this surgery, which allows total stromal replacement of the recipient's cornea, leaving an intact recipient Descemet membrane and endothelium. Along with the advances in surgical instrumentation, improvements in surgical techniques and imaging technology have contributed to improved visual outcomes obtained with DALK. DALK is now seen as a viable alternative to penetrating keratoplasty with equivalent visual results and better long-term graft survival.

A survey of contact lens complications in Singapore.

OBJECTIVES: To describe the type of complications related to contact lens wear seen in a hospital setting in Singapore. METHODS: Data were collated over a 2-year period from April 1999 to March 2001, from all public hospitals in Singapore. A standardized clinical record form was completed by the attending doctor when a patient presented for contact lens-related complications. RESULTS: Nine hundred and fifty-three contact lens-related complications were recorded. The mean age of presentation was 26.4 years. Women made up the majority n=491 (68%). Most patients were Chinese (555, 77%). Six hundred and seventy-six (93.7%) patients were soft contact lens wearers. Most patients, 585 patients (85.2%) wore their lenses on a daily basis. Two hundred and forty-four cases (25.6%) of infective keratitis were seen, with 55 patients requiring hospital admission for management of the infection. Soft disposable contact lens wear was associated with the majority of the infective keratitis (178, 73%). Epithelial keratitis occurred in 229 patients (24.0%) with punctuate epithelial erosions being the commonest presentation in 139 (14.6%). Allergic conjunctivitis occurred in 179 (18.8%) of the cases with giant papillary conjunctivitis accounting for 147 (15.4%) of them. Dry eyes (77, 8.1%), sterile infiltrates (71, 7.5%) and neovascularization of the cornea (75, 7.9%) were the next commonest complications seen. Corneal edema, solution- and lens-related complications occurred in small numbers, accounting for less than 5% of the complications reported. CONCLUSION: Infective keratitis secondary to soft lens wear was the most common complication, followed by epithelial keratitis and allergic conjunctivitis seen in public hospitals in Singapore.