Topical Spironolactone in the Treatment of Evaporative Dry Eye Disease.

Meibomian gland dysfunction (MGD) is associated with evaporative dry eye syndrome, which is characterized by a reduction in meibum secretion and tear film instability. Present treatments provide only temporary relief, thereby necessitating the exploration of novel therapeutic strategies for chronic treatment. This study aims to evaluate topical spironolactone, a medication with anti-mineralocorticoid, anti-androgenic, and anti-inflammatory properties, in treating dry eye. A retrospective observational study was performed on the medical records of 102 patients diagnosed with dry eye disease. These patients were categorized into two groups based on their Schirmer's tear test scores. Various clinical indicators, including subjective global assessment scores, visual acuity, keratitis, conjunctival staining scores, and lid margin health, were evaluated prior to and following treatment with topical spironolactone eye drops. The group with higher Schirmer's scores exhibited improvement in self-reported global assessment scores after treatment. Significant improvements were also observed in keratitis and conjunctival staining scores, visual acuity, and lid margin inflammation. Similarly, the group with lower Schirmer's scores demonstrated improvements in self-reported global assessment scores and visual acuity after treatment. Topical spironolactone may improve tear film quality and address the inflammatory processes associated with MGD and evaporative dry eye. Moreover, the topical administration of spironolactone in an ocular vehicle appears to be well tolerated and may mitigate the risk of systemic adverse effects. Further studies are warranted to explore the long-term effects of topical spironolactone in the treatment of evaporative dry eye disease.

Topical Spironolactone in the Treatment of Ocular Graft-Versus-Host Disease.

INTRODUCTION: This two-part study aimed to investigate the therapeutic potential of topical spironolactone in ocular graft-versus-host disease (oGVHD). While off-label use of topical spironolactone has been described in dry eye, its efficacy in managing signs and symptoms of oGVHD remains unstudied. Preclinically, we tested the hypothesis that spironolactone induces corneal lipid synthesis in a mouse model. Clinically, we assessed patient response to spironolactone with a retrospective observational design. METHODS: Both immortalized and primary human corneal epithelial cells were stained with oil red O after 9 days of treatment with spironolactone. C57BL/6 mice were dosed thrice daily with one drop in each eye for 18 days. Corneal tissue was stained with oil red O and BODIPY™. Twenty eyes with oGVHD, as defined by the International Chronic oGVHD Consensus Group, were studied. Corneal fluorescein staining, lid margin vascularity, meibomian gland obstruction, meibum turbidity, zone A posterior lid margin vascularity, and oGVHD diagnostic criteria severity grading were compared in a pre-post study. Follow-up times ranged from 7 to 21 weeks, with a median time of 12 weeks. Statistical analysis was done with STATA 17 by fitting data to a non-parametric model. RESULTS:  In vitro results showed an increased number and density of oil red O staining granules in the treatment group versus control in both primary and immortalized human corneal epithelium. In vivo, results showed translation to the mouse model with increased corneal epithelial BODIPY™ signal compared to untreated control. oGVHD patients had improved lid margin vascularity (p = 0.046), corneal fluorescein staining (p = 0.021), and International oGVHD Consensus Group severity scores (p = 0.011) after treatment with topical spironolactone. Minimal adverse effects were noted, the most common being mild stinging lasting less than a minute after instillation. CONCLUSION: The improved severity scores, lid margin inflammation, and corneal fluorescein staining after weeks of treatment support the rationale that topical spironolactone may benefit oGVHD. The observed lipid production by the corneal epithelium is thought to contribute to this protective effect against ocular surface erosive disease in oGVHD. A mineralocorticoid receptor antagonist, spironolactone may offer therapeutic benefits in oGVHD while avoiding undesirable side effects of topical or systemic glucocorticoids.

Changes in preoperative corneal measurements following same-day intraocular pressure testing with rebound tonometry.

PURPOSE: To evaluate the extent to which rebound tonometry affects corneal surface properties and preoperative corneal measurements. SETTING: Four cornea specialty private practices. DESIGN: Prospective case series. METHODS: Visual acuity testing, corneal topography, keratometry, and grading of corneal staining were performed on both eyes of 60 randomly selected, previously scheduled patients. Technicians then performed rebound tonometry on one randomly selected eye only. Immediately following, intraocular pressure measurement, corneal topography, keratometry, and corneal staining were repeated on both eyes. RESULTS: None of the 60 study eyes developed increased staining scores following intraocular pressure testing with the Icare ic100. For corneal staining, mean keratometry, and total corneal cylinder, no statistically significant difference was found from the first measurement to the second measurement between the study eyes and control eyes. CONCLUSION: Rebound tonometry with the Icare ic100 may be used on any patient at any time during the exam without affecting the results of other tests, allowing clinicians to test intraocular pressure prior to preoperative cataract or refractive surgery measurements on the same day. This may allow for significant improvement in patient flow in the office and save patients from the cost and time of extra visits.

A novel mutation of the Keratin 12 gene responsible for a severe phenotype of Meesmann's corneal dystrophy.

PURPOSE: To determine if a mutation within the coding region of the keratin 12 gene (KRT12) is responsible for a severe form of Meesmann's corneal dystrophy. METHODS: A family with clinically identified Meesmann's corneal dystrophy was recruited and studied. Electron microscopy was performed on scrapings of corneal epithelial cells from the proband. Mutations in the KRT12 gene were sought using direct genomic sequencing of leukocyte DNA from two affected and two unaffected family members. Subsequently, the observed mutation was screened in all available family members using polymerase chain reaction and direct sequencing. RESULTS: A heterozygous missense mutation (Arg430Pro) was found in exon 6 of KRT12 in all 14 affected individuals studied. Unaffected family members and 100 normal controls were negative for this mutation. CONCLUSIONS: We have identified a novel mutation in the KRT12 gene that is associated with a symptomatic phenotype of Meesmann's corneal dystrophy. This mutation results in a substitution of proline for arginine in the helix termination motif that may disrupt the normal helix, leading to a dramatic structural change of the keratin 12 protein.

Spontaneous and inheritable R555Q mutation in the TGFBI/BIGH3 gene in two unrelated families exhibiting Bowman's layer corneal dystrophy.

PURPOSE: Bowman's layer corneal dystrophies (CDBs) include 2 distinct types: CDB1, or Reis-Bücklers (RBCD), and CDB2, or Thiel-Behnke (TBCD). We studied the genetic basis of 2 cases of apparent spontaneous CDB mutations and attempted to determine if these are sporadic and inheritable mutations. DESIGN: Retrospective molecular genetic study and case report. PARTICIPANTS: Twelve patients were recruited from 2 unrelated families for this study, including 2 affected individuals from one family (family A) and 1 affected individual from another (family B). METHODS: Slit-lamp examination was performed for each patient to determine the disease phenotype. Histological analysis of affected cornea specimens was used for identification of pathogenic corneal opacities in 2 affected patients from family A. MAIN OUTCOME MEASURES: Genomic DNA was isolated from the blood samples and used for mutation screening of the TGFBI/BIGH3 gene. Sixteen polymorphic DNA markers from 9 different chromosomes were used to establish the maternity and paternity of the 2 probands. RESULTS: The 2 families were confirmed to be unrelated. The age onset of ocular symptoms was <2 years for all 3 affected patients. Clinical diagnoses of CDB1 (RBCD) and CDB2 (TBCD) were made for probands A and B, respectively. The affected corneas showed epithelial haze with diffuse, irregular, patchy opacities in a honeycomb and geographic pattern. Subepithelial plaques, increased trichome staining of anterior stroma, and irregular Bowman's layer were observed. An R555Q mutation was found in TGFBI/BIGH3 in the 2 probands but not in their parents. The son of proband A was also affected and apparently inherited his disease allele from his father. CONCLUSION: The R555Q mutation occurred spontaneously and independently in the 2 unrelated CDB families and was confirmed to be transmitted to the next generation in 1 of the 2 families. These findings strongly support the notion that a genetic diagnosis should be determined for CDB and other dystrophies associated with mutations in TGFBI/BIGH3. The discovery of a spontaneous mutation should alert clinicians to be aware of the existence of genetic alterations for their patients without apparent family history of the disease.

Excimer laser exacerbation of Avellino corneal dystrophy.

We review the clinical, histopathological, and ultrastructural findings and DNA phenotyping of a patient with Avellino corneal dystrophy exacerbated by laser in situ keratomileusis. The findings are reported and interpreted in the context of a literature review. The case highlights the possible difficulty of recognizing subtle dystrophic findings, as well as the importance of avoiding refractive surgical intervention in patients with Avellino corneal dystrophy to avoid exacerbation of dystrophic deposits in the cornea and subsequent reduction in vision.

Isolation of the ocular surface to treat dysfunctional tear syndrome associated with computer use.

Dysfunctional tear syndrome (DTS) associated with computer use is characterized by mild irritation, itching, redness, and intermittent tearing after extended staring. It frequently involves foreign body or sandy sensation, blurring of vision, and fatigue, worsening especially at the end of the day. We undertook a study to determine the effectiveness of periocular isolation using microenvironment glasses (MEGS) alone and in combination with artificial tears in alleviating the symptoms and signs of dry eye related to computer use. At the same time, we evaluated the relative ability of a battery of clinical tests for dry eye to distinguish dry eyes from normal eyes in heavy computer users. Forty adult subjects who used computers 3 hours or more per day were divided into dry eye sufferers and controls based on their scores on the Ocular Surface Disease Index (OSDI). Baseline scores were recorded and ocular surface assessments were made. On four subsequent visits, the subjects played a computer game for 30 minutes in a controlled environment, during which one of four treatment conditions were applied, in random order, to each subject: 1) no treatment, 2) artificial tears, 3) MEGS, and 4) artificial tears combined with MEGS. Immediately after each session, subjects were tested on: a subjective comfort questionnaire, tear breakup time (TBUT), fluorescein staining, lissamine green staining, and conjunctival injection. In this study, a significant correlation was found between cumulative lifetime computer use and ocular surface disorder, as measured by the standardized OSDI index. The experimental and control subjects were significantly different (P<0.05) in the meibomian gland assessment and TBUT; they were consistently different in fluorescein and lissamine green staining, but with P>0.05. Isolation of the ocular surface alone produced significant improvements in comfort scores and TBUT and a consistent trend of improvement in fluorescein staining and lissamine green staining. Isolation plus tears produced a significant improvement in lissamine green staining. The subjective comfort inventory and the TBUT test were most effective in distinguishing between the treatments used. Computer users with ocular surface complaints should have a detailed ocular surface examination and, if symptomatic, they can be effectively treated with isolation of the ocular surface, artificial tears therapy, and effective environmental manipulations.

The zebrafish cornea: structure and development.

PURPOSE: To evaluate the zebrafish as a model for the studies of corneal development and disease. METHODS: Zebrafish embryos and larvae at various stages of development were used for documenting corneal morphogenesis and differentiation. Corneal samples were collected from embryos, larvae, and adult zebrafish for histologic and electron microscopy analysis. Expression patterns of corneal polypeptides were investigated by immunostaining of sections. RESULTS: The zebrafish cornea develops rapidly during embryogenesis, so that its three major layers, the epithelium, the stroma, and the endothelium, are well formed by day 3 postfertilization. The subsequent steps of corneal differentiation, such as the thickening of the corneal stroma, proceed relatively slowly. Several polypeptides are highly enriched in the epithelium or the stroma of the larval and adult zebrafish cornea and are excellent markers of corneal differentiation. CONCLUSIONS: Development and differentiation of the zebrafish cornea are easily accessible to analysis. Anatomic and ultrastructural characterization of the zebrafish cornea demonstrates many similarities to the human cornea and provides the basis for the use of the zebrafish model both to analyze the basic genetic mechanisms of corneal development and to study the causes of corneal disease.

Comparison of the relative toxicity of travoprost 0.004% without benzalkonium chloride and latanoprost 0.005% in an immortalized human cornea epithelial cell culture system.

The purpose of this study was to compare the relative toxicity of a new topical ophthalmic glaucoma medication, travoprost 0.004% without benzalkonium chloride (BAK), with that of commercially available latanoprost 0.005% (preserved with 0.02% BAK) in immortalized human corneal epithelial cells (HCEs). Tissue culture plates (96 well) containing HCEs were divided into 6 groups. Two groups served as negative controls (70% methanol and gentamicin). Another 2 groups--1 in corneal epithelial culture media and the other in a hydroxypropyl (HP)-Guargellable lubricant eyedrop--served as live controls. The travoprost 0.004% without BAK and latanoprost 0.005% groups were exposed to 100 microL of the undiluted solutions. Cells were incubated for 25 min at 37 degree C. A live/dead assay was used to measure the effects of travoprost without BAK and of latanoprost on HCEs compared to 70% methanol and culture medium. Between the 2 glaucoma medications tested, travoprost 0.004% preserved without BAK showed significantly less toxicity on HCEs than did latanoprost 0.005%. This difference may have ramifications in terms of tolerability for patients who use these topical glaucoma drugs on a long-term basis.

Whole Exome Sequencing and Segregation Analysis Confirms That a Mutation in COL17A1 Is the Cause of Epithelial Recurrent Erosion Dystrophy in a Large Dominant Pedigree Previously Mapped to Chromosome 10q23-q24.

PURPOSE: To report identification of a COL17A1 mutation in a family with a corneal dystrophy previously mapped to chromosome 10q23-q24. METHODS: Whole-exome sequencing was performed on DNA samples from five affected family members and two unrelated, unaffected individuals. Identified variants were filtered for those that were: located in the linked interval on chromosome 10q23-q24; novel or rare (minor allele frequency ≤0.01); heterozygous; present in all affected individuals and not in controls; and present in genes that encode proteins expressed in human corneal epithelial cells (reads per kilobase per million ≥1). Sanger sequencing of identified variants (SNVs) was performed in additional family members. In silico analysis was used to predict the functional impact of non-synonymous variants. RESULTS: Three SNVs located in two genes were identified that met the filtering criteria: one rare synonymous c.3156C>T variant in the collagen, type XVII, alpha I (COL17A1) gene; and two rare variants, one synonymous and one missense, in the dynamin binding protein (DNMBP) gene. Sanger sequencing of additional family members determined that only the COL17A1 variant segregates with the affected phenotype. In silico analysis predicts that the missense variant in DNMBP would be tolerated. CONCLUSIONS: The corneal dystrophy mapped to chromosome 10q23-q24 is associated with the c.3156C>T variant in COL17A1. As this variant has recently been identified in five other families with early onset recurrent corneal erosions, and has been shown in vitro to introduce a cryptic splice donor site, this dystrophy is likely caused by aberrant splicing of COL17A1 and should be classified as epithelial recurrent erosion dystrophy.

Computer vision syndrome: a review.

As computers become part of our everyday life, more and more people are experiencing a variety of ocular symptoms related to computer use. These include eyestrain, tired eyes, irritation, redness, blurred vision, and double vision, collectively referred to as computer vision syndrome. This article describes both the characteristics and treatment modalities that are available at this time. Computer vision syndrome symptoms may be the cause of ocular (ocular-surface abnormalities or accommodative spasms) and/or extraocular (ergonomic) etiologies. However, the major contributor to computer vision syndrome symptoms by far appears to be dry eye. The visual effects of various display characteristics such as lighting, glare, display quality, refresh rates, and radiation are also discussed. Treatment requires a multidirectional approach combining ocular therapy with adjustment of the workstation. Proper lighting, anti-glare filters, ergonomic positioning of computer monitor and regular work breaks may help improve visual comfort. Lubricating eye drops and special computer glasses help relieve ocular surface-related symptoms. More work needs to be done to specifically define the processes that cause computer vision syndrome and to develop and improve effective treatments that successfully address these causes.

A randomized, investigator- masked clinical trial comparing the efficacy and safety of gatifloxacin 0.3% administered BID versus QID for the treatment BID versus QID for the treatment of acute bacterial conjunctivitis of acute bacterial conjunctivitis.

PURPOSE: To compare the efficacy and safety of To compare the efficacy and safety of gatifloxacin ophthalmic solution 0.3% (Zymar) administered BID versus QID in patients with acute bacterial conjunctivitis. METHODS: In a randomized, investigator-masked clinical trial, patients diagnosed with bacterial conjunctivitis (based on signs and symptoms) received gatifloxacin either BID or QID for 5 days. Visits were scheduled at day 0, day 3, and day 5. Conjunctival cultures were taken at each visit. The clinical cure rate at day 5 was determined for the entire patient population (primary endpoint). Additionally, clinical cure at day 5 was evaluated for a population of patients defined a priori (per protocol) as being culture positive at baseline and with no substantial protocol deviations. Safety was determined through recording of adverse events. Minimal inhibitory concentrations (MIC) and susceptibility of isolates to gatifloxacin were determined using a broth dilution method. RESULTS: Patient characteristics in both the BID and QID groups (N = 104) were similar in terms NN of baseline demographics and disposition. The clinical cure rate on day 5 in the entire, intentto-treat (ITT) population was 86.5% (45/52) in the gatifloxacin BID group and 71.2% (37/52) in the gatifloxacin QID group (95% CI: [-0.03, 30.80]; p = 0.096). In both treatment groups, 5/52 patients (9.6%) reported adverse events. The most common adverse event was conjunctivitis. No serious adverse events were reported. In the a priori-defined per-protocol (PP) population, the clinical cure rate on day 5 was 95.5% (21/22) in the gatifloxacin BID group and 85.7% (18/21) in the gatifloxacin QID group (95% CI: [-7.57, 21.05]; p = 0.294). At baseline, 96.1% (98/102) of the isolates were susceptible to gatifloxacin. The overall MIC(90) (mean +/- standard error of the mean) was 0.5 +/- 1.3 microg/mL. CONCLUSION: In this study, gatifloxacin 0.3% administered BID was as effective and as safe as gatifloxacin 0.3% administered QID for 5 days for the treatment of bacterial conjunctivitis.

Recurrence of chromosome 10 Thiel-Behnke corneal dystrophy (CDB2) after excimer laser phototherapeutic keratectomy or penetrating keratoplasty.

PURPOSE: To evaluate the recurrence of Thiel-Behnke dystrophy (linked to the 10 q23-q24 locus) after phototherapeutic keratectomy or penetrating keratoplasty. METHODS: This is a retrospective study of 4 patients (8 eyes) who underwent phototherapeutic keratectomy and 1 patient (2 eyes) who underwent penetrating keratoplasty. Best corrected visual acuity was assessed, and biomicroscopic examinations for evidence of recurrent dystrophy were documented and photographed. The location, lesion distribution, and lesion pattern of any recurrence was noted. RESULTS: Follow-up ranged from 8 months to 25 years (mean +/- SD 9.7 +/- 7.97 years). All 10 eyes showed biomicroscopic evidence of central recurrence. Six eyes showed an intermediate zone of honeycomb opacities as well as a peripheral zone of focal and geographic lesions. Despite the high incidence of recurrence, functional central visual acuity was maintained. All eyes maintained functional best corrected visual acuity (ranging from 20/25 to 20/80) despite the postoperative recurrence. CONCLUSION: Recurrence of Thiel-Behnke corneal dystrophy is extremely high after either phototherapeutic keratectomy or penetrating keratoplasty. Despite the high incidence of recurrence, the central cornea is the last to be affected. The peripheral-to-central progression of the lesions points to an epithelial origin for the pathogenesis of the dystrophy. Phototherapeutic keratectomy in the treatment of Thiel-Behnke corneal dystrophy offers a safe and effective treatment modality, providing patients up to 8 years of improved vision ranging from 8 months to 8 years (mean +/- SD 3.7 +/- 2.7 years) and delaying or circumventing the need for more invasive intraocular surgical intervention.

Electron microscopy of surface smoothness of porcine corneas and acrylic plates with four brands of excimer laser.

PURPOSE: This study compares ablation smoothness patterns produced on four different excimer laser devices available for photorefractive surgery. METHODS: VISX calibration plastic and porcine cornea were ablated with standard -3.00-D, -6.00-D, and -9.00-D settings using four different excimer lasers: VISX S2 Smooth Scan, Nidek EC-5000, Autonomous Ladar Vision System, and Bausch and Lomb Technolas. Electron microscopy and laser interferometry were used for qualitative evaluation of the ablated surfaces. Corneal ablation surface smoothness was graded by ten independent observers. Calibration plastic ablated surfaces were evaluated quantitatively for smoothness by laser interferometry. RESULTS: The independent observer assessment of corneal ablation surface smoothness demonstrated that the Autonomous small spot Gaussian profile laser produced the smoothest ablation surfaces, followed by the other broad beam lasers. In comparing ablation smoothness among various refractive powers (-9.00 D, -6.00 D, and -3.00 D), a trend was observed that indicated a correlation of higher refractive settings with decreasing surface smoothness. However, this trend was not statistically significant. The quantitative laser interferometry measurements supported the independent observer ranking of the Autonomous flying small spot ablation profile as the smoothest. However, there were differences between the laser interferometry smoothness rankings and independent observer smoothness rankings. CONCLUSION: There were significant differences in ablation surface smoothness among the four excimer lasers tested.

Laser epithelial keratomileusis for myopia with the autonomous laser.

PURPOSE: To describe the refractive outcome, objective clinical data, and subjective patient experiences after laser epithelial keratomileusis (LASEK) at 1, 3, and 6 months after surgery. METHODS: This was a retrospective, nonrandomized, comparative study of 58 LASEK-treated eyes (36 patients) with myopia (with and without astigmatism) between -1.50 and -14.75 D (mean -7.80 +/- 2.90 D, median -7.90 D). Refractive surgery was performed using the Alcon Summit Autonomous LADAR Vision excimer laser. Manifest refraction, best-spectacle and uncorrected Snellen visual acuity, stability of refraction, and corneal haze were evaluated before surgery and up to 6 months after surgery. A group of randomly selected LASIK-treated eyes were compared at each time point. RESULTS: Patients who opted for monovision (n=12) were excluded. In the emmetropia targeted eyes (n=46), 45%, 83%, 85%, and 89% achieved 20/40 or better uncorrected Snellen visual acuity (UCVA) at 1 day, 1 week, 2 weeks, and 1 month respectively. At 6 months, 73% (n=28) of eyes treated achieved UCVA 20/20 with 97% achieving 20/40 or better (mean, -0.51 D). At 3 and 6 months, 71% (n=46) and 68% (n=28) were within +/- 0.50 D of emmetropia. The percentage of eyes that achieved UCVA 20/40 or better at 6 months was 97% (n=28). Visually significant corneal haze was evident in two LASEK-treated patients (four eyes) at 6 months. No eyes lost two or more lines of best spectacle-corrected Snellen visual acuity. CONCLUSIONS: Preliminary data suggest that LASEK appears to be a safe, effective, and comparable alternative to LASIK, even for higher amounts of myopia. A prospective, randomized clinical trial is needed to better define the role of LASEK as it compares to other refractive procedures, specifically LASIK and PRK.

Prediction of corneal haze using an ablation depth/corneal thickness ratio after laser epithelial keratomileusis.

PURPOSE: To investigate the usefulness of ablation depth/corneal thickness (AD/CT) ratio to predict corneal haze after laser epithelial keratomileusis (LASEK) using a retrospective, comparative, interventional case series. METHODS: Fifty patients (90 eyes; mean age 40.9 years) with myopia, hyperopia, and/or astigmatism underwent bilateral or unilateral LASEK for correction of refractive error. After epithelial flaps were created using an 18% alcohol solution, bilateral or unilateral LASEK was performed using the Alcon Autonomous LADARVision 4000 excimer laser. Visual acuity (best spectacle-corrected and uncorrected) and refractive error were measured before and after LASEK. Corneas were assessed by two independent evaluators under a slit-lamp biomicroscope with broad tangential illumination. The relative haze scale was quantitated: 0 (clear), 0.5+ (trace), 1+ (mild), 2+ (moderate), 3+ (marked), and 4+ (severe). RESULTS: Mean preoperative spherical equivalent refraction was -5.46 +/- 3.74 D (range -12.375 to +5.00 D), mean ablation depth was 93.04 +/- 45.03 microm (range 21.2 to 207.2 microm), and mean AD/CT ratio was 0.18 +/- 0.09 (range 0.04 to 0.41). Of 90 eyes, 40 eyes had a higher ablation depth (AD/CT ratio > 0.18) and 50 eyes had a lower ablation depth (AD/CT ratio < 0.18); 92.5% of eyes in the higher ratio group developed clinically significant haze (1+ or greater). In the lower ratio group, 94% of eyes developed no more than 1+ corneal haze, if any. CONCLUSION: The ablation depth/corneal thickness ratio is useful for predicting corneal haze after LASEK. An AD/CT ratio of 0.18 or more suggests that patients have a high risk of developing clinically significant haze (1+ or more) after LASEK.

Cleavage of corneal basement membrane components by ethanol exposure in laser-assisted subepithelial keratectomy.

PURPOSE: To determine the anatomic cleavage plane after exposure to 20% ethanol for approximately 20 to 25 seconds to create an epithelial flap in laser-assisted subepithelial keratectomy (LASEK). SETTING: Ocular Surface Research & Education Foundation, Miami, Florida, and Hermann Eye Center Refractive Surgery Center, Houston, Texas, USA. METHODS: Immunofluorescence staining using monoclonal antibodies against laminin 5, collagen VII, and integrins beta(1) and beta(4) was performed to determine the anatomic location of the cleavage plane in an epithelial flap created by 20-second exposure to 20% ethanol in cadaver eyes and in epithelial flaps obtained from LASEK patients. RESULTS: Immunofluorescence staining to laminin 5 and integrin beta(4) was patchy in the lifted flap and the remaining corneal basement membrane. Immunostaining to collagen VII, the main component of anchoring fibrils, remained exclusively in the corneal bed. Immunostaining to integrin beta(1), present in the pericellular location of all epithelial cell layers, remained exclusively in the epithelial flap. This finding was consistent in cadaver corneas and LASEK epithelial flaps. CONCLUSIONS: The cleavage plane of the ethanol-induced corneal epithelial flap is located between the lamina lucida and the lamina densa of the basement membrane, where integrin beta(4) interacts with laminin 5 to form hemidesmosomes.

Exclusion of the human collagen type XVII (COL17A1) gene as the cause of Thiel-Behnke corneal dystrophy (CDB2) on chromosome 10q23-q25.

PURPOSE: Determination of the gene causing Thiel-Behnke Corneal Dystrophy (CDB2) would have important clinical implications. Previous studies in our laboratory have suggested that the COL17A1 gene may be the cause of Thiel-Behnke Corneal Dystrophy (CDB2) on Chromosome 10q23-q25. METHODS: We evaluated a five-generation family with CDB2 mapped to chromosome 10. Many of these family members were diagnosed by slit-lamp microscopy. In addition, genomic DNA was isolated and purified from peripheral blood samples. The COL17A1 gene was screened for possible disease causing mutations by PCR and DNA sequencing analysis. RESULTS: No disease-causing mutations were found in any of the 56 exons of the COL17A1 gene or in any of the flanking intron/exon junctions. CONCLUSIONS: Mutations in the coding sequence of the human collagen XVII (COL17A1) gene are not the cause of CDB2.

Bis(zinc-dipicolylamine), Zn-DPA, a new marker for apoptosis.

PURPOSE: To characterize the labeling of apoptotic cells with a molecular probe of bis(zinc(II)-dipicolylamine) (Zn-DPA) conjugated with a fluorescent reporter in a rat model of retinal ganglion cell (RGC) degeneration induced by N-methyl-D-aspartate (NMDA). METHODS: Adult Wistar rats were given unilateral intravitreal injections of 3 µL 40 mM neutralized NMDA and euthanized at 1, 2, 4, 24, and 48 hours. One hour before euthanasia, 3 µL Zn-DPA conjugated with fluorescein (Zn-DPA 480) was intravitreally injected. Prelabeling of RGC with retrograde fluorogold (FG), TUNEL, and immunohistochemistry with III ß-tubulin and vimentin were performed. RESULTS: Fluorescence labeling of Zn-DPA 480 was observed in the retinas from 1 hour up to 24 hours after NMDA injection, whereas the labeling was reduced at 48 hours postinjection. At both 4 and 24 hours postinjection, most Zn-DPA 480-positive cells in the RGC layer were labeled by FG and III ß-tubulin. The number of TUNEL-positive cells increased from 4 to 24 hours. At 24 hours, 95.7% of Zn-DPA 480-positive cells were TUNEL positive, whereas 95.1% of TUNEL-positive cells were Zn-DPA 480 positive. The numbers of Zn-DPA 480-positive cells at 1 and 2 hours after NMDA injection were significantly higher than TUNEL. CONCLUSIONS: Our findings demonstrate that intravitreal injection of fluorescent Zn-DPA 480 labels retinal neurons undergoing apoptosis and that recognition of exposed phosphatidylserine appears earlier than detection of DNA fragmentation, indicating the potential of Zn-DPA as an imaging probe for tracking degenerating retinal neurons.