Strengthening of the barrier function in human telomerase reverse transcription (hTERT) immortalized corneal and conjunctival epithelium by double-stranded RNA.

To investigate the response to polyinosinic:polycytidylic acid [poly(I:C)], a double-stranded RNA Toll-like receptor 3 agonist that mimics viral infection, in the barrier function of two established human telomerase reverse transcriptase-immortalized cell lines, termed HCLE for the human corneal-limbal epithelial line and HCjE for the human conjunctival-epithelial line. In this study, HCLE and HCjE cells were used to evaluate the underlying mechanism of epithelial-cell barrier function regulation. Briefly, HCLE and HCjE cells were first cultured on 12-well Transwell® (Corning®) filter-plates, and reverse transcription-polymerase chain reaction, western blotting, and immunohistochemical examinations were then performed to assess tight junction (TJ)-related protein expression and cellular distribution. Next, the barrier function of the cells was measured via transepithelial electrical resistance (TEER) and paracellular molecular flux. The cells were then stimulated with poly(I:C) and the TEER and TJ-related protein expressions were analyzed. Similar to that in in vivo epithelium, the expression of claudin (CLDN) subtypes CLDN-1, -4, and -7 was observed in the HCLE and HCjE cells, and the barrier function in the HCLE cells was tighter than that in the HCjE cells. Post stimulation with poly(I:C), TEER of the HCLE and HCjE cells increased in a dose- and time-dependent manner, the production of TJ-related protein mRNA and CLDN-4 protein were elevated, and the barrier function of the HCLE and HCjE cells increased, thus possibly indicating that the increased barrier function is a defense mechanism against viral infection.

Toxicity of Amphotericin B in Rabbit Corneal Epithelial Cells Stored in Optisol™-GS: Corneal Epithelial Cell Morphology and Migration.

PURPOSE: To evaluate the toxicity of Amphotericin B (AmB) in Optisol™-GS Corneal Storage Media (Bausch & Lomb) on corneal epithelial cell (CEC) morphology and migration ability. METHODS: Sclerocorneal strips were removed from male Japanese white rabbits, and then stored at 4 °C in Optisol™-GS containing 0 µg/ml of AmB (control group) and 2.5, 5, 25, and 50 µg/ml of AmB (AmB groups; four eyes per group). After 7 days of storage, CEC morphology was evaluated by hematoxylin-eosin staining, immunohistochemical staining (ZO-1), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Moreover, to evaluate CEC migration ability, three corneal blocks (6-8 × 3 mm each) from one preserved cornea were cultured for 24 h, and the area of CEC migration (2 mm at the central region) onto the stromal surface was then measured. RESULTS: At 5, 25, and 50 µg/ml of AmB, deformation and vacuolation of CECs were observed in all preserved corneas. ZO-1 expression was significantly reduced in corneas preserved at AmB concentrations of 25 and 50 µg/ml. TUNEL Labeling Index was significantly increased at AmB concentrations of ≥5 µg/ml. CEC migration was inhibited in a dose-dependent manner at AmB concentrations of 25 and 50 µg/ml compared to the control group. CONCLUSIONS: The addition of AmB to Optisol™-GS can be toxic to CECs and inhibit their migration at a concentration of ≥5 µg/ml. AmB at a concentration of 2.5 µg/ml can be considered safe for the preservation of donor corneal tissue used in corneal epithelial transplantation surgery.

Tight junction transmembrane protein claudin subtype expression and distribution in human corneal and conjunctival epithelium.

PURPOSE: The combination of the tight junction transmembrane protein claudin subtypes is one of the most important determinants of variations in the tightness of individual paired tight junction strands. The barrier function of corneal epithelium is much stronger than that of conjunctival epithelium. In this study, the expression and cellular distribution of claudin species in in vivo human corneal and conjunctival epithelium were investigated. METHODS: Reverse transcription-polymerase chain reaction was used to reveal the claudin mRNA. Immunohistochemistry was used to determine the tissue distribution of tight junction-related proteins and MUC5AC. RESULTS: Transcripts for claudin-1, -2, -3, -4, -7, -9, and -14 were identified in human corneal epithelium. Transcripts for claudin-1, -2, -4, -7, -9, -10, and -14 were identified in human conjunctival epithelium. By immunohistochemistry, claudin-1, -4, and -7 were found to be localized at the membrane of human corneal and conjunctival epithelial cells. In human conjunctival epithelium, claudin-10 staining was observed at several, but not all, apical epithelial cell-to-goblet cell junctions. CONCLUSIONS: Claudin-1, -4, and -7 are expressed in corneal and conjunctival epithelia. Claudin-10 is prominent at several junctions between apical epithelial cells and goblet cells in conjunctival epithelium. Except for claudin-10 expression in conjunctival epithelium, the claudin subtype expressions of corneal and conjunctival epithelia are similar. Therefore, there must be a difference between these two epithelial types with regard to the specific ratio of claudin subtypes expressed or their phosphorylation status. The distribution of goblet cells in conjunctival epithelium also influences the difference in barrier function.

Cultivated corneal endothelial transplantation in a primate: possible future clinical application in corneal endothelial regenerative medicine.

PURPOSE: To review our attempt to devise a method of cultivated corneal endothelial transplantation using primates in which corneal endothelium, like that of humans, has low proliferative ability. METHODS: Monkey corneal endothelial cells (MCECs) were cultivated, with subcultures grown on collagen type I carriers. The corneal endothelia of 6 eyes of 6 monkeys were scraped intensively, after which cultivated MCEC sheets were inserted into the anterior chamber of 4 eyes. As controls, a collagen sheet without MCECs was transplanted in 1 eye of a monkey, and a suspension of cultivated MCECs was injected into the anterior chamber of 1 eye of another monkey. RESULTS: MCECs produced a confluent monolayer of closely attached hexagonal cells, which expressed both ZO-1 and Na-K adenosine triphosphatase. Early postoperative period MCEC sheets were attached to Descemet membrane, and corneal clarity was recovered. Six months after transplantation, MCEC-transplanted corneas remained clear, and closely attached hexagonal cells were observed. In 1 animal with longer observation, polygonal cells were observed by in vivo specular microscopy at a density of >2000 cells/mm2 and remained >1600 cells/mm2 for < or =2 years. Control eyes showed irreversible bullous keratopathy throughout the observation period. CONCLUSIONS: Cultivated MCECs become attached to the transplanted eye and maintain a clear cornea < or =2 years postoperatively, suggesting that corneal endothelial cells of primates might have proliferative ability in vivo once they have been cultured and proliferated in vitro. Our monkey model constitutes an important step forward for regenerative medicine with possible future application in patients with corneal endothelial dysfunction.

Cultivated corneal endothelial cell sheet transplantation in a primate model.

PURPOSE: To examine the feasibility of cultivated corneal endothelial cell transplantation in a primate model. METHODS: Monkey corneal endothelial cells (MCECs) obtained from three cynomolgus monkeys were cultivated, with subcultures grown on collagen type I carriers for 4 weeks. The corneal endothelium of the right eye of six monkeys was mechanically scraped, after which a cultivated MCEC sheet was brought into the anterior chamber of four eyes and fixed to Descemet's membrane by air. As the control, a collagen sheet without MCECs was transplanted into one eye of one monkey, and a suspension of cultivated MCECs was injected into the anterior chamber in one eye. RESULTS: Cultivated MCECs produced a confluent monolayer of closely attached hexagonal cells that showed both ZO-1 and Na(+)-K(+) ATPase expression. In the early postoperative period MCEC sheets were attached to Descemet's membrane and corneal clarity was recovered. The recovered clarity was accompanied by a decrease in corneal thickness. Fluorescein DiI labeled donor corneal endothelial cells were detected on the host cornea on postoperative day 7. Six months after transplantation MCEC-transplanted corneas remained clear, and hexagonal cells were observed by in vivo specular microscopy with a density of 1992 to 2475 cells/mm(2). Control eyes showed irreversible bullous keratopathy that precluded pachymetry and specular microscopy. CONCLUSIONS: A model of cultivated corneal endothelial transplantation for corneal endothelial dysfunction was established in primates whose corneal endothelial cells have less proliferative capacity in vivo. Our results suggest that this is a useful model for long-term observation in advance of the future clinical application of cultivated corneal endothelial transplantation.

Phenotypic investigation of cell junction-related proteins in gelatinous drop-like corneal dystrophy.

PURPOSE: To identify the molecules involved in the pathogenesis of gelatinous drop-like corneal dystrophy (GDLD) by using immunohistochemical analysis of the expression of tight junction (TJ)-, desmosome-, and basement membrane (BM)-related proteins in human corneas with GDLD. METHODS: Mutation analysis was performed on samples from three Japanese women with GDLD. Four corneal buttons from these patients were examined histopathologically by Congo red staining and immunohistochemical analysis for the expression of TJ-related proteins (ZO-1, occludin, and claudin-1), desmosome components (desmoplakin), BM-related proteins (integrins alpha6, beta4, alpha3, and beta1; laminin-5; and collagens IV and VII), amyloid P component, and lactoferrin. RESULTS: Mutation analysis revealed that all three women had the Q118X mutation on M1S1. There were accumulations, primarily beneath the epithelium, of Congo-red-positive deposits with birefringence under polarized light. The BM was abnormally thickened and demonstrated a bandlike area. Immunofluorescence analysis revealed that neither ZO-1 nor occludin was expressed in the TJ areas of surface epithelial cells; there was no expression of claudin-1 or desmoplakin in the epithelial surface layer of GDLD corneas. Integrins alpha6, beta4, alpha3, and beta1 was expressed along the serrated surface of the BM, laminin-5 and collagens IV and VII were widely expressed throughout the BM, and lactoferrin was expressed in the amyloid deposits and the thickened BM. CONCLUSIONS: This is the first demonstration of the unique expression patterns of the major cell-junction-related proteins in GDLD corneas. The results show that in corneas with the Q118X mutation, there is a disturbance in cell-to-cell and cell-to-substrate junctions. These findings are an important step toward elucidating the pathogenesis of GDLD.

Intraocular pressure change during hemodialysis.

PURPOSE: To evaluate intraocular pressure (IOP) change through hemodialysis and examine other factors to affect IOP change in patients treated with hemodialysis. DESIGN: Prospective, observational case series. METHODS: IOP, blood pressure, and plasma colloid osmotic pressure (COP) were measured in 188 eyes of 95 patients receiving hemodialysis in Nantan General Hospital. RESULTS: IOP significantly decreased after hemodialysis (P = 0.006). Body weight, systolic and diastolic blood pressure also decreased significantly (P < 0.001). COP significantly increased (P < 0.001). Change in IOP correlated negatively with COP change (OD; r = -0.196, OS; r = -0.339) and positively with change in body weight (OD; r = -0.278, OS; r = -0.357). IOP tended to increase after hemodialysis in patients receiving hemodialysis for more than 12 years. CONCLUSIONS: IOP decrease during hemodialysis may be due to COP increase. Patients receiving hemodialysis for a long time are more likely to show IOP increase after hemodialysis.

[The usefulness of frequency doubling technology perimetry in glaucoma screening in health-check program].

PURPOSE: Visual field testing has not been used as a screening procedure because it requires too much testing time and manpower. We evaluated the usefulness of Frequency Doubling Technology(FDT) visual field testing as a screening procedure for glaucoma in a health-check screening program. METHODS: A total of 800 eyes of 400 persons were examined for visual acuity, noncontact tonometry, slit-lamp biomicroscopy, funduscopy, and FDT testing of visual fields (N-30-5). The initial screening result was considered positive for glaucoma if any abnormality of FDT perimetry was detected, the intraocular pressure exceeded 21 mmHg, or funduscopy showed glaucomatous disc cupping or retinal nerve fiber layer defect. The re-examination comprised several ophthalmic examinations such as automatic perimetry 9 Humphrey field analyzer). RESULTS: Glaucoma was suspected in 118 eyes; 16 eyes were thus diagnosed after re-examination. FDT detected visual field abnormalities in 15 eyes. There were 40 eyes that were not diagnosed although FDT detected visual field abnormalities. CONCLUSIONS: FDT detected visual field abnormalities in glaucoma patients with high sensitivity and specificity. FDT is a useful screening test for glaucoma.

Tight junction-related protein expression and distribution in human corneal epithelium.

PURPOSE: To investigate the expression and cellular distribution of the tight junction-related proteins occludin, claudin and ZO-1 in human corneal epithelium. METHODS: Light and electron immunohistochemistry was used to determine tissue distribution of occludin, claudin-1 and ZO-1 in the human corneal epithelium. Reverse transcription-polymerase chain reaction was used to reveal claudin mRNA expression in human corneal epithelium. RESULTS: In transverse sections, occludin and ZO-1 were localized at the apical cell-cell junctions between superficial cells in stratified corneal epithelium. Both basal and basolateral membranes of superficial cells were stained by the claudin-1 antibody, but no apical membrane staining was observed. In en face sections, claudin-1 and ZO-1 antibodies showed as bands that corresponded to the junctional complex. Claudin-1 staining of superficial cell cytoplasm was also observed. Occludin staining was seen at the junctional complex, where it was not continuous, but dotted along the cell junctions. The transcripts for claudin-1 and several other claudin isotypes, such as -2, -3, -4, -7, -9 and -14 were identified. CONCLUSION: Not only occludin, but also some claudins act as integral transmembrane proteins in the corneal epithelium. ZO-1 is a component of the corneal epithelial tight junction, as it is in most epithelial cells.

Comparison of ultrastructure, tight junction-related protein expression and barrier function of human corneal epithelial cells cultivated on amniotic membrane with and without air-lifting.

PURPOSE: To evaluate the usefulness of the air-lifting technique for culturing corneal limbal epithelial cells on amniotic membrane (AM) for use in ocular surface reconstruction. A cultured sheet that has a good barrier function should be better for this purpose. In corneal epithelium, tight junctions (TJ) play a vital role in the barrier function. The TJ complex includes the integral transmembrane proteins occludin and the claudins, and some membrane-associated proteins such as ZO-1. In this paper, we investigated the barrier function and the expression of TJ related proteins. METHODS: Corneal limbal epithelium obtained from donor corneas and cultivated on acellular AM was divided into two groups. These were the non-air-lifting (Non-AL) group, which was continuously submerged in medium, and the air-lifting (AL) group, which was submerged in medium for 3 weeks, then exposed to air by lowering the medium level. Morphology and the permeability to horseradish peroxidase (HRP) were determined by electron microscopy. Tight junction (TJ)-related protein and mRNA expression changes were assessed by immunoblotting and reverse transcription-polymerase chain reaction. RESULTS: The cultures of both groups formed 4-5-layer-thick, well-stratified epithelium. The AL cultures had tightly packed epithelial cells with all the HRP/diaminobenzidine (DAB) reaction product accumulated on the apical surface of the superficial cells. The Non-AL culture, by contrast, had more loosely packed epithelial cells with larger intercellular spaces. The HRP/DAB reaction product penetrated the intercellular space to a depth of 3-4 cell layers. Statistically, there was a significant difference in intercellular spaces and desmosome count in the superficial cells between the groups. With AL, TJ-related proteins localized at the apical portion of the lateral membrane. TJ-related protein and mRNA amounts were not changed by AL while claudin subtype expression became more consistent and closer to that of in vivo corneal epithelium. CONCLUSIONS: The AL technique reduces intercellular spaces in the superficial cells and promotes the formation of the barrier function. It is useful in culturing corneal epithelial cells for use in ocular surface reconstruction.