Yoko Akune

Role of Insulin in Regulation of Na-/K-Dependent ATPase Activity and Pump Function in Corneal Endothelial Cells

PURPOSE The Na(+)-/K(+)-dependent ATPase (Na,K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. The role of insulin in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells was investigated. METHODS Confluent monolayers of mouse corneal endothelial cells were exposed to insulin. ATPase activity was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate; Na,K-ATPase activity was defined as the portion of total ATPase activity sensitive to ouabain. Pump function was measured with the use of a Ussing chamber; pump function attributable to Na,K-ATPase activity was defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis and immunocytochemistry were performed to measure the expression of the Na,K-ATPase alpha(1)-subunit. RESULTS Insulin increased the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were blocked by protein kinase C (PKC) inhibitors and protein phosphatases 1 and 2A inhibitor. Western blot analysis indicated that insulin decreased the ratio of the inactive Na,K-ATPase alpha(1)-subunit. Immunocytochemistry indicated that insulin increased the cell surface expression of the Na,K-ATPase alpha(1)-subunit. CONCLUSIONS These results suggest that insulin increases the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. The effect of insulin is mediated by PKC and presumably results in the activation of PP1, 2A, or both, which are essential for activating Na,K-ATPase by alpha(1)-subunit dephosphorylation.

Diquafosol Tetrasodium Increases the Concentration of Mucin-like Substances in Tears of Healthy Human Subjects

Abstract Purpose: This study was undertaken to determine the effect of topical application of diquafosol tetrasodium on proteins and mucin-like substances from tears of clinically healthy subjects. Methods: Tears were collected from both the eyes of 10 healthy volunteers. Diquafosol tetrasodium solution (3%) was applied once to the right eye and 0.9% sodium chloride solution (saline) once to the left eye. Tear samples were collected by Schirmer test strips before application and 5, 15, 30 and 60 min after application. Sialic acid, a marker of mucin-like substances, and major tear proteins including secretory IgA, lactoferrin, lipocalin-1, and lysozyme were measured by high performance liquid chromatography. Results: Levels of total protein, sIgA and lysozyme were transiently decreased in both groups but returned to baseline levels within 15 min after application. The concentration of lactoferrin and lipocalin-1 did not change significantly in both groups. Sialic acid in tears was significantly decreased 5 min after saline application, but significantly increased 5 min after diquafosol application. No significant difference in sialic acid was seen after 15 min in both groups. Conclusions: Topical application of saline and diquafosol resulted in transient decrease of tear proteins possibly due to wash out or dilution effects. In contrast, diquafosol application significantly increased sialic acid, although the effect was transient. This suggests diquafosol stimulates the secretion of mucins from ocular tissues of healthy human subjects.

Biochemical analyses of lipids deposited on silicone hydrogel lenses

Purpose This study was performed to determine the levels of lipids deposited on in vivo worn silicone hydrogel lenses.

Cost-utility Analysis of Screening for Diabetic Retinopathy in Japan: A Probabilistic Markov Modeling Study

ABSTRACT Purpose: To evaluate the cost-effectiveness for a screening interval longer than 1 year detecting diabetic retinopathy (DR) through the estimation of incremental costs per quality-adjusted life year (QALY) based on the best available clinical data in Japan. Methods: A Markov model with a probabilistic cohort analysis was framed to calculate incremental costs per QALY gained by implementing a screening program detecting DR in Japan. A 1-year cycle length and population size of 50,000 with a 50-year time horizon (age 40–90 years) was used. Best available clinical data from publications and national surveillance data was used, and a model was designed including current diagnosis and management of DR with corresponding visual outcomes. One-way and probabilistic sensitivity analyses were performed considering uncertainties in the parameters. Results: In the base-case analysis, the strategy with a screening program resulted in an incremental cost of 5,147 Japanese yen (¥; US

Diquafosol for Soft Contact Lens Dryness: Clinical Evaluation and Tear Analysis.

64.6) and incremental effectiveness of 0.0054 QALYs per person screened. The incremental cost-effectiveness ratio was ¥944,981 (US

Fluorophotometric Analysis of the Ocular Surface Glycocalyx in Soft Contact Lens Wearers

11,857) per QALY. The simulation suggested that screening would result in a significant reduction in blindness in people aged 40 years or over (−16%). Sensitivity analyses suggested that in order to achieve both reductions in blindness and cost-effectiveness in Japan, the screening program should screen those aged 53–84 years, at intervals of 3 years or less. Conclusions: An eye screening program in Japan would be cost-effective in detecting DR and preventing blindness from DR, even allowing for the uncertainties in estimates of costs, utility, and current management of DR.

The Clinical Effectiveness and Cost-Effectiveness of Screening for Age-Related Macular Degeneration in Japan: A Markov Modeling Study

Purpose To evaluate the efficacy of topical application of 3% diquafosol tetrasodium solution for the treatment of soft contact lens (SCL) wearers with dryness. In addition to clinical tests and subjective symptoms, we assessed the fluorescence intensity of wheat germ agglutinin conjugate of fluorescein (F-WGA) used as a marker of membrane-associated mucins and sialic acid concentration in tear fluids as a marker of secreted mucins. Methods Twelve SCL wearers with dryness symptoms were treated with diquafosol for 4 weeks. Clinical tests included the tear film break-up time (BUT), corneal and conjunctival fluorescein staining scores, and Schirmer values. Subjective symptoms were evaluated by the Dry Eye-Related Quality-of-Life Score (DEQS). Fluorescence intensities in the central cornea were measured by fluorophotometry at 5 minutes after a 5% F-WGA solution was applied to the eye. The tears collected by the Schirmer test strips were analyzed by high-performance liquid chromatography (HPLC), and the concentrations of sialic acid; total protein; and the four major tear proteins secretory IgA, lactoferrin, lipocalin-1, and lysozyme proteins were measured. Results Comparing the results before and after diquafosol treatment, BUT (p < 0.01), kerato-conjunctival staining score (p < 0.05), corneal staining score (p < 0.05), and DEQS score (p < 0.01) showed statistically significant improvements. The F-WGA fluorescence intensities (p < 0.0001) significantly increased after treatment, whereas the concentrations of sialic acid and tear proteins remained unchanged. Conclusions Topical application of diquafosol solution to the SCL wearers with dryness improved biomarker of membrane-associated mucins, BUT, staining of cornea and conjunctiva, and subjective symptoms.

Evaluation of the frequency of ophthalmic solution application: washout effects of topical saline application on tear components.

Abstract Purpose: Unstable tear film characterized by shorting of tear break-up time (BUT) is associated with discomfort and dryness in contact lens wearers. The glycocalyx is thought to be crucial in maintaining the wettability and lubrication of the ocular surface. We evaluated the ocular surface glycocalyx in soft contact lens (SCL) wearers using a fluorescein-labeled wheat germ agglutinin (F-WGA) as a marker to demonstrate the ocular surface glycoconjugates in vivo. Methods: Twenty experienced SCL wearers and 20 healthy volunteers with no history of CL wearing (controls) were enrolled in the study. After applying a 5% F-WGA solution to the eyes of study individuals, fluorescent intensities in their respective central corneas were measured by fluorophotometry. The relationship between F-WGA intensity in the corneal surface and clinical parameters associated with contact lens wear were analyzed. Results: F-WGA fluorescence intensity in the SCL group was 418.5 ± 103.3, which was significantly lower than that of the controls (825.0 ± 179.8; p < 0.0001, Mann–Whitney test). F-WGA fluorescence intensity was not correlated with Schirmer’s test values or age, whereas a statistically significant correlation between F-WGA fluorescence intensity and tear film BUT was observed (r = 0.77, p < 0.0001). The decrease in F-WGA fluorescence intensity could be reversed by discontinuation of SCL use. Conclusion: Reduction and/or compositional alteration of ocular surface glycocalyx may be one of the causative factors of SCL-induced eye dryness.

Influence of Ophthalmic Solutions on Tear Components.

Objective To investigate the cost-effectiveness of screening and subsequent intervention for age-related macular degeneration (AMD) in Japan. Methods The clinical effectiveness and cost-effectiveness of screening and subsequent intervention for AMD were assessed using a Markov model. The Markov model simulation began at the age of 40 years and concluded at the age of 90 years. The first-eye and second-eye combined model assumed an annual state-transition probability, development of prodromal symptoms, choroidal neovascularization (CNV), and reduction in visual acuity. Anti–vascular-endothelial-growth-factor (anti-VEGF) intravitreal injection therapy and photodynamic therapy (PDT) were performed to treat CNV. Intake of supplements was recommended to patients who had prodromal symptoms and unilateral AMD. Data on prevalence, morbidity, transition probability, utility value of each AMD patient, and treatment costs were obtained from published clinical reports. Results In the base-case analysis, screening for AMD every 5 years, beginning at the age of 50 years, showed a decrease of 41% in the total number of blind patients. The screening program reduced the incidence of blindness more than did the additional intake of supplements. However, the incremental cost-effectiveness ratio (ICER) of screening versus no screening was 27,486,352 Japanese yen (JPY), or 259,942 US dollars (USD) per quality-adjusted life year (QALY). In the sensitivity analysis, prodromal symptom-related factors for AMD had great impacts on the cost-effectiveness of screening. The lowest ICER obtained from the best scenario was 4,913,717 JPY (46,470 USD) per QALY, which was approximately equal to the willingness to pay in Japan. Conclusions Ophthalmologic screening for AMD in adults is highly effective in reducing the number of patients with blindness but not cost-effective as demonstrated by a Markov model based on clinical data from Japan.

The effect of rebamipide ophthalmic suspension on ocular surface mucins in soft contact lens wearers

Abstract Purpose: To determine and compare the effects of single and frequent topical applications of saline solution on tear protein concentration in clinically normal subjects. Materials and methods: Tears were collected from both eyes of 11 normal volunteers using Schirmer’s strips. Saline solution (0.9% sodium chloride) was applied once in the right eye and five times with an interval of 1 min each in the left eye. Tears were collected before and 5, 15, 30 and 60 min after application of the solution. Total tear protein concentration in the samples was measured by the Bradford method and major tear protein concentration (secretory immunoglobulin A, lactoferrin, lipocalin-1, lysozyme and sialic acid) was measured using a high-performance liquid chromatography assay. Results: A significant decrease was observed in the concentration of total tear protein, major tear proteins and sialic acid after topical application of saline solution. This decrease was attributed to the washout and dilution effect. A low protein concentration persisted longer with more frequent application of the solution. The concentration of secretory immunoglobulin A and sialic acid concentration recovered slowly compared with that of other proteins. Conclusions: Even a single application of saline solution resulted in significant changes in major tear protein and sialic acid concentration in the tears of normal subjects. Differences in the recovery of tear protein concentration may be related to the process of protein production and secretion. A balance between normal tear function and the therapeutic effects of ophthalmic solutions should be considered when deciding the frequency of application, particularly in patients with dry eye.