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Purpose: Considering that tears play the role of a lubricant, it is speculated that in the pathophysiology of dry eye, increased friction during blinking results in corneal and conjunctival damage, which may subsequently affect the blink. The purpose of this study was to investigate the relationship between ocular surface epithelial damage, tear abnormalities, and blinks in patients with dry eye., Methods: This study involved 45 eyes of 45 female patients with dry eye (mean age: 57.6 years). In all eyes, tear meniscus radius (mm), spread grade of the tear film lipid layer (SG: 1-5: 1 being the best), fluorescein breakup time (FBUT, seconds), corneal and bulbar conjunctival epithelial damage (CED: 15 points maximum and CONJUNCTIVAL EPITHELIAL DAMAGE (CjED): 6 points maximum, respectively), and Schirmer I test (ST1, mm) were evaluated. Blink rate (BR, blinks per minute), palpebral aperture height (mm), upper-eyelid opening-phase amplitude/upper-eyelid closing-phase amplitude (mm), upper-eyelid opening-phase duration/upper-eyelid closing-phase duration (ms), and upper-eyelid opening-phase maximum velocity/upper-eyelid closing-phase maximum velocity (mm/s) were measured using a custom-made high-speed blink analyzer. Finally, the factors that determine CED and CjED were investigated by multiple regression analysis, in which the parameters were chosen using the stepwise procedure., Results: CED and CjED were found to be described as 2.687 + (1.816 × SG) - (0.937 × FBUT) (R = 0.656, P < 0.0001) and 0.684 + (0.801 × SG) - (0.526 × FBUT) - (0.041 × ST1) + (0.010 × upper-eyelid closing-phase maximum velocity) (R = 0.714, P < 0.0001), respectively., Conclusions: Although CED was significantly related to only tear abnormalities, CjED was significantly related to tear abnormalities and blinking.

Dry eye syndrome (DES), a multifactorial disease of the tears and ocular surface, is one of the most common ocular disorders. Tear film contains ocular mucins and is essential for maintaining the homeostasis of the wet ocular surface. Since there are a limited number of clinical options for the treatment of DES, additional novel treatments are needed to improve the clinical results. In this study, we found that placental extract-derived dipeptide (JBP485) clearly promoted the expression and secretion of gel-forming mucin 5ac (Muc5ac) in rabbit conjunctival epithelium. JBP485 also elevated the expression level of cell surface-associated mucins (Muc1/4/16) in rabbit corneal epithelium. The Schirmer tear test results indicated that JBP485 induced tear secretion in the rabbit model. Moreover, JBP485 clinically improved corneal epithelial damage in a mouse dry eye model. Thus, our data indicate that JBP485 efficiently promoted mucin and aqueous tear secretion in rabbit ocular surface epithelium and has the potential to be used as a novel treatment for DES.

Purpose: To perform a surface chemistry study of the interactions between benzalkonium chloride (BAC), a common preservative used in ophthalmic formulations, and tear film (TF) constituents., Methods: The interactions between BAC and human tears, meibum, and rabbit corneal cell lipid extracts at the air-water interface were examined in vitro during controlled compression-expansion of the film area by a Langmuir surface balance, surface potential measurements, and pendant drop-axisymmetric drop shape analysis (PD-ADSA). Surface pressure-area isotherms and isocycles were used to assess the sample's lateral elasticity and capability of compressing and spreading during dynamic area changes. Lipid film morphology was monitored by Brewster angle microscopy. The viability of BAC-treated Statens Seruminstitut rabbit cornea (SIRC) cell cultures was also examined. The BAC concentration was kept within the clinical range of 0.001% to 0.02%., Results: In the Langmuir balance and PD-ADSA experiments, the interactions between BAC and lipids or tears resulted in (1) impaired lipid spread and formation of discontinuous nonuniform surface layers, (2) increased surface pressure-area hysteresis during compression and expansion, and (3) displacement of the lipids by BAC from the surface. A decrease (>50%) in SIRC cell viability was observed. The effects occurred within seconds after BAC exposure, and their magnitude increased with BAC concentration., Conclusions: The surface chemistry approach used in this study provided molecular-scale insights into the detrimental effect of BAC on TF, which well explain the TF instability and corneal epithelial barrier dysfunction after exposure to BAC in the in vivo human eye.

Purpose: To date, no studies have elucidated the composition of the corneal filament in detail. In this study, an immunohistochemical technique was used to clarify the exact composition of the corneal filament in filamentary keratitis. In addition, the mechanisms responsible for filament formation were identified., Methods: Filaments were obtained from 13 patients with filamentary keratitis with a background of penetrating keratoplasty, aqueous tear deficiency, and severe ocular surface disorders, who were receiving treatment at an outpatient facility. From those tissues, transverse and longitudinal frozen sections were prepared and subjected to an indirect fluorescent immunohistochemical analysis with primary antibodies, including cytokeratins (CK1, -4, -6, -10, -12, and -13), mucins (MUC1, -4, -5AC, and -16), keratinization-related proteins (transglutaminase [TGase]-1 and filaggrin), cell proliferation marker Ki67, and markers of infiltration cells (HLA-DR and neutrophil-elastase). TUNEL staining was used for the detection of apoptosis. Fluorescent images of the sections were inspected with a fluorescence microscope., Results: The filaments were composed of CK12-positive cells and had a roll-formed central core. They were covered with MUC5AC- and -16-positive mucins including CK4- and -13-positive cells and neutrophil-elastase-positive cells. The filaments also included broken cells and DNA fiber-form postlesional nuclei that were positive for TUNEL staining. However, those areas stained weakly for CK6 and HLA-DR; faintly for CK1, CK10, MUC1, and MUC4; and not at all for Ki67, TGase-1, and filaggrin., Conclusions: The results of this research have the potential to open new pathways toward understanding the mechanism that generates the filament in filamentary keratitis, as well as new treatments in the future.

The aim of this study was to evaluate the efficacy and safety of trehalose-treated freeze-dried amniotic membrane (TT-FDAM) for ocular surface reconstruction. Human AM deprived of amniotic epithelial cells was first incubated with 10% trehalose solution, and then freeze-dried, vacuum-packed, and sterilized with gamma-irradiation. The resultant newly developed TT-FDAM was characterized for its physical, biological, and morphological properties by comprehensive physical assays, immunohistochemistry, electron microscopy, cell adhesion assay, 3D cell culture, and an in vivo biocompatibility test. The adaptability of TT-FDAM was markedly improved as compared to FDAM. Immunohistochemistry for several extracellular matrix molecules revealed that the process of freeze-drying and irradiation apparently did not affect its biological properties, however, electron microscopy revealed that the detailed morphological appearance of TT-FDAM is more similar to that of native AM than to FDAM. Intracorneal and scleral-surface transplantation of TT-FDAM showed excellent biocompatibility with ocular surface tissues. Thus, TT-FDAM retained most of the physical, biological, and morphological characteristics of native AM, consequently it is a useful biomaterial for ocular surface reconstruction.

Purpose: To evaluate the use of autologous serum (AS)-derived cultivated corneal epithelial transplantation for the treatment of severe ocular surface disease., Design: Retrospective noncomparative case series., Participants: Nine eyes from 9 patients with total limbal stem cell deficiency were studied. These consisted of 2 eyes with Stevens-Johnson syndrome, 1 with chemical injury, 1 with ocular cicatricial pemphigoid, 1 with Salzmann corneal dystrophy, 1 with aniridia, 1 with graft-versus-host disease, and 2 with idiopathic ocular surface disease., Methods: Autologous serum obtained from patients was used for cultivating corneal epithelial cells on an amniotic membrane substrate. These AS-derived corneal epithelial equivalents were compared with those derived from fetal bovine serum (FBS)-supplemented medium. At the time of surgery, complete removal of the corneal pannus and conjunctiva up to 3 mm from the limbus was performed. Allogeneic (7 cases) and autologous (2 cases) AS-derived cultivated corneal epithelial equivalents were transplanted onto the ocular surface. Postoperative follow-up included serial slit-lamp examinations with fluorescein staining, as well as photographic documentation., Main Outcome Measures: Ocular surface reconstruction with corneal epithelialization, graft integrity, visual acuity, and postoperative complications., Results: The corneal epithelial sheets cultivated in AS- and FBS-supplemented media were morphologically similar, and demonstrated the normal expression of tissue-specific keratins and junctional specialization assembly proteins. After transplantation, complete corneal epithelialization was achieved within 2 to 5 days. All eyes demonstrated an improvement in visual acuity by > or =2 lines. During the follow-up period of 14.6+/-4.36 months, the corneal surface of all patients remained stable and transparent, without significant complications., Conclusions: Transplantation of AS-derived cultivated corneal epithelial equivalents was shown to be a feasible method of treating patients with severe ocular surface disease. The use of AS is of clinical importance in the development of autologous xenobiotic-free bioengineered ocular surface equivalents for clinical transplantation.

Purpose: To compare the effects of levobunolol hydrochloride and timolol maleate on tear volume, precorneal tear film stability, and corneal epithelial barrier function in normal human eyes., Subjects and Methods: The study population consisted of 14 healthy volunteers. To obtain pretreatment baseline values, we determined the radius of the tear meniscus (RTM) by meniscometry; the noninvasive breakup time (NIBUT) of the precorneal tear film with a tear specular microscope; and corneal fluorescein uptake with a fluorophotometer. Levobunolol hydrochloride (0.5%) or timolol maleate solution (0.5%) was instilled twice daily for 4 weeks into 1 eye; the contralateral eye was treated with the other topical drug twice daily for the same period. At the end of the study period, the same tests were performed, and the pre- and posttreatment results were compared., Results: Timolol solution did, and levobunolol did not, significantly reduce NIBUT from the baseline values. RTM was significantly decreased by treatment with either timolol or levobunolol solution. Corneal fluorescein uptake was not significantly changed, although it was higher after treatment with both topical drugs., Conclusions: Four-week treatment with timolol solution resulted in significant instability of the precorneal tear film. Both timolol and levobunolol solution significantly decreased tear volume on the ocular surface. These results indicate that levobunolol solution applied twice daily has equal effects on the tear volume and corneal epithelial barrier function as does timolol solution applied twice daily and that it affects precorneal tear film stability less than timolol solution.

Aim: To evaluate the influence of topically administered latanoprost, a prostaglandin F(2alpha) analog, on the corneal epithelial barrier function., Patients and Methods: Twenty-four patients suffering from a glaucoma were enrolled. Ten patients without prior topical antiglaucoma medication received topical latanoprost (0.005%, once daily) for 30 days (monotherapy group); 14 patients receiving topical antiglaucoma medication also received latanoprost (0.005%, once daily) for 180 days (combination treatment group). Before and 30 days after treatment (monotherapy group) and before and 30 and 180 days after treatment (combination group), the corneal epithelial barrier function was measured by a fluorophotometric technique., Results: The fluorescein uptakes in the monotherapy group were 54.6 +/- (SE) 7.5 and 57.1 +/- 11.0 ng/ml before and 30 days after treatment, respectively (p = 0.81). In the combination group, the uptakes were 101.0 +/- 18.3 and 118.9 +/- 25.9 ng/ml (p = 0.38) and 93.4 +/- 17.5 ng/ml (p = 0.58) before and 30 and 180 days after treatment, respectively., Conclusion: The corneal epithelial barrier function remained intact following the instillation of latanoprost in both groups., (Copyright 2002 S. Karger AG, Basel)

An appreciation of the biological characteristics of the human ocular surface epithelium affords us a great insight into the physiology of the human ocular surface in health and disease. Here, we review five important aspects of the human ocular surface epithelium. First, we recognize the discovery of corneal epithelial stem cells, and note how the palisades of Vogt have been suggested as a clinical marker of their presence. Second, we introduce the concept of the gene expression profile of the ocular surface epithelium as arrived at using a new strategy for the systematic analysis of active genes. We also provide a summary of several genes abundantly or uniquely expressed in the human corneal epithelium, namely clusterin, keratin 3, keratin 12, aldehyde dehydrogenase 3 (ALDH3), troponin-I fast-twitch isoform, ssig-h3, cathepsin L2 (cathepsin V), uroplakin Ib, and Ca(2+)-activated chloride channel. Genes related to limbal and conjunctival epithelia are also described. Third, we touch upon the genetic abnormalities thought to be involved with epithelial dysfunction in Meesmann's dystrophy, gelatinous drop-like corneal dystrophy, and the ssig-h3-mutated corneal dystrophies. Fourth, we provide an update regarding the current state of knowledge of the role of cytokines, growth factors and apoptosis in relation to ocular surface homeostasis and tissue reconstruction; the main factors being epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), transforming growth factor-ss (TGF-ss), and some inflammatory cytokines. Fifth, corneal epithelial barrier function and dysfunction as measured by fluorophotometry is remarked upon, with an explanation of the FL-500 fluorophotometer and its ability to detect corneal epithelial dysfunction at a subclinical level. The research described in this review has undoubtedly generated a complete understanding of corneal epithelial pathophysiology-an understanding that, directly or indirectly, has helped advance the development of new therapeutic modalities for ocular surface reconstruction.

Purpose: We evaluated the effect on corneal epithelium barrier function of instillation of prostaglandin F2 alpha ophthalmic solution (latanoprost) for one month., Materials and Methods: Ten healthy volunteers and nine glaucoma patients were enrolled in this study. The barrier function was determined as uptake of topically applied sodium fluorescein by the central cornea measured with an anterior fluorophotometer(FL-500, Kowa Co. Ltd). Healthy volunteers and glaucoma patients received 0.005% latanoprost instillation once daily for one month. We measured the uptake of fluorescein by the cornea of each subject before and one month after instillation., Results: Fluorescein uptake before the instillation was 22.2 +/- 16.0 ng/ml (mean +/- standard deviation) and 26.4 +/- 15.1 ng/ml one month after the treatment in the normal group, and it was 55.0 +/- 25.0 ng/ml before treatment and 57.8 +/- 37.0 ng/ml after treatment in the glaucoma group. There was no significant difference in the uptake of fluorescein before and after treatment in either of two groups., Conclusion: These results suggested that the barrier function of corneal epithelium was not compromised after the instillation of latanoprost for at least one month.

Purpose: We investigated the adverse effects of beta-blocker eye drops on tears and ocular surface epithelium., Methods: We studied twenty-three eyes of twenty three glaucoma patients [10 males, 13 females: 53.8 +/- 12.2 (yrs; mean +/- standard deviation)] treated with beta-blocker eye drops for more than three months and thirty two control subjects (16 males, 16 females: 50.4 +/- 10.9). The parameters described below were compared between the groups: 1. the radius of tear meniscus curvature, 2. grades for tear lipid layer interference patterns, 3. non-invasive breakup time (N-BUT), 4. cotton thread value, 5. scores of fluorescein staining, 6. fluorescein breakup time (F-BUT), 7. scores of rose bengal staining, 8. and Schirmer I value., Results: The glaucoma group showed a significant decrease in the radius of tear meniscus curvature (p = 0.0007), a significantly lower distribution in the grades for tear lipid layer interference patterns (p = 0.0270), a significant difference in the scores of fluorescein staining (p < 0.0001), a significant shortening in F-BUT (p = 0.0050), a significantly higher distribution in the scores of rose Bengal staining (p = 0.0010), and a significantly smaller value in Schirmer I value (p = 0.0042). However, there was no significant difference in N-BUT and cotton thread value., Conclusions: These results clearly demonstrate that the ocular surface in glaucoma patients treated with beta-blocker eye drops show dry-eye-like changes in terms of tears and ocular surface epithelium.

Purpose: To report two cases with corneal epithelial disturbance during topical use of the new prostaglandin F 2 alpha derivative latanoprost and a beta-blocker., Cases: Both case 1 (82-year-old male) and case 2 (50-year-old female) were treated with topical antiglaucoma drugs for ocular hypertension., Findings: One month (case 1) and 10 days (case 2) after treatment with topical beta-blocker and latanoprost, corneal epithelial disturbance occurred. The corneal epithelial disturbance improved over several weeks with the preservative-free artificial tears after discontinuation of latanoprost and the beta-blocker., Conclusion: Careful observation is necessary during the course of treatment with latanoprost and a beta-blocker.

To investigate the long-term effect of a topically applied beta-blocker on human corneal epithelium, the corneal epithelial barrier function and the superficial cell area of the corneal epithelium were evaluated. Seventeen normal healthy volunteers (without medication), 7 cataract patients (treated with pyrenoxine eyedrops) and 7 glaucoma or ocular hypertension patients (treated with 0.5% timolol maleate) were assigned to this study. The eyedrops had been used on a daily basis for at least 3 months. In the evaluation of corneal epithelial barrier function, fluorescein uptake was measured using a slitlamp fluorophotometer after application of 3 microl of 0.5% fluorescein for 10 min. In the evaluation of the superficial cell area, the central corneal epithelium was measured by tandem scanning confocal microscopy (TSCM). The healthy control and timolol groups were compared. Corneal fluorescein uptake in the healthy control, pyrenoxine and timolol groups was 20.3 +/- 3.2, 21.5 +/- 4.0 and 76.2 +/- 30.0 ng/ml (mean +/- standard error), respectively. There was a significantly higher fluorescein uptake in the timolol group compared to the pyrenoxine group (p = 0.0088) and the healthy control group (p = 0.0055). TSCM showed no significant difference in the superficial cell areas of the corneal epithelium between the healthy control and timolol groups. beta-Blocker eyedrops decreased the corneal epithelial barrier function. Their application was not accompanied by any biomicroscopic change in the superficial cell area., (Copyright 2000 S. Karger AG, Basel)

Purpose: To confirm the usefulness of fluorescein dextran as a vital dye for observing corneal epithelial disturbance through a medical-use soft contact lens., Methods: Ten percent fluorescein isothiocyanate-dextran (FITC-dextran) was used as the staining dye. Patients with corneal epithelial disturbance who were wearing therapeutic soft contact lenses were observed using FITC-dextran., Results: Through the soft contact lens, FITC-dextran enabled corneal disturbance observation similar to that seen with sodium fluorescein. In a case of corneal perforation, as well, positive Seidel's test was confirmed through the soft contact lens using this technique., Conclusion: FITC-dextran is considered a useful dye for examining the corneal epithelial condition without contact lens removal.

Purpose: The effect of overnight wear of graded rigid gas-permeable (RGP) contact lenses on the rabbit corneal epithelial barrier function was assessed by fluorophotometry., Methods: RGP lenses with optimal fitting were worn by rabbits for 24 h. The values of oxygen transmissibility (Dk/L) of lenses used were 0, 10, 27, 43, 84, and 109 x 10(-9) (cm/s)(ml O2/ml mm Hg); lens thickness (0.15 mm) and diameter (14.0 mm) were standardized. Corneal epithelial barrier function was measured by a slit-lamp fluorophotometer. Corneal surface lesions by fluorescein staining were observed by a slit-lamp biomicroscope with the aid of fluorescein filter paper and were scored. Corneal epithelial structure was observed by scanning electron microscopy (SEM)., Results: After 24-h wear of RGP lenses, fluorescein uptake by the cornea increased, and this increase depended on the Dk/L value of lenses worn; mean +/- standard error, 1,601+/-454 (Dk/L 0), 347+/-226 (Dk/L 10), 144+/-42 (Dk/L 27), 100+/-25 (Dk/L 43), 56+/-18 (Dk/L 84), 51+/-15 (Dk/L 109), and 49+/-4 ng/ml (control). There was a significant correlation between the fluorophotometric value and the biomicroscopic scores of corneal lesions. SEM showed progressive desquamation of epithelial superficial and wing cells for lower-value Dk/L lens groups., Conclusion: This study demonstrated for the first time that overnight wear of Dk/L 84 and 109 lenses does not alter the barrier function of the rabbit corneal epithelium. In contrast, wear of a PMMA lens greatly alters the corneal epithelial barrier function. It is suggested that RGP lenses with a Dk/L >80 can provide safe overnight wear, based on the epithelial barrier function.

Aims: To assess the integrity of the ocular surface epithelium in patients with atopic dermatitis from the viewpoint of its barrier function., Methods: 49 patients with atopic dermatitis with blepharoconjunctivitis (ABC group), 27 age matched patients with seasonal or perennial allergic conjunctivitis (AC group), and 20 volunteers with normal healthy eyes (NH group) were assigned to this study. Ocular surface epithelium barrier function was evaluated by the fluorophotometric method using a slit lamp fluorophotometer. 3 microliters of 0.5% sodium fluorescein was instilled into the conjunctival sac of the right eye and fluorescein uptake (ng/ml) 30 minutes later (20 minutes after eye washing) was measured in the central cornea and the temporal bulbar conjunctiva. Fluorophoto metric measurements performed were analysed in each group and compared between the groups., Results: The ABC group showed significantly higher fluorescein uptake (mean 28.2 (SEM 3.3) ng/ml) in the cornea than the AC (11.4 (2.2), p = 0.001) and NH groups (9.3 (2.1), p = 0.001). There was no significant difference between the AC and NH groups (p = 0.930). The ABC group also showed significantly higher fluorescein uptake in the bulbar conjunctiva (393.4 (54.0)) than the AC (182.9 (24.6), p = 0.011) and NH groups (169.3 (29.1), p = 0.012). There was also no significant difference in fluorescein uptake between the AC group and the NH group (p = 0.987)., Conclusion: This study suggested that ocular surface epithelium barrier function is impaired in patients with atopic dermatitis with blepharoconjunctivitis.

The effect of concomitant administration of galactose and the aldose reductase inhibitor(ARI) Epalrestat (Kinedak) on corneal barrier function was examined in dogs. Six-month-old male beagles were rendered aphakic in one eye and then divided into four groups as follows: 1) a control group fed on 30% cellulose, 2) a galactosemic group fed on 30% galactose, 3) a 30% galactose-fed group treated with low dose (20 mg/kg) ARI and 4) a 30% galactose-fed group treated with high dose (50 mg/kg) ARI. Forty-one months after the start of these diets, corneal autofluorescence and the corneal barrier function were measured in each dog using anterior fluorophotometry (FL-500). When barrier function was analyzed in non-operated eyes, fluorescence data were significantly higher in the galactosemic group compared to the control group. In non-operated eyes, fluorescent data in high-dose ARI treated group were significantly lower than those in the galactosemic group. However, in operated eyes, no significant difference was observed between the galactosemic group and the ARI treated groups. Similar trends were observed when corneal autofluorescence of each group was compared. Long-term galactose feeding appeared to damage corneal epithelial barrier function. This damage was not observed in the high-dose ARI treated group suggesting that this damage may be linked to the polyol pathway.