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Purpose The purpose of the present study was to investigate the role of glutathione peroxidase 4 (GPx4) in conjunctival epithelial cells. Methods An immortalized human conjunctival epithelial cell line was used. Cells were transfected with catalase, GPx1, GPx4, SOD1, SOD2, or control siRNA. Knockdown was confirmed by RT-PCR and immunoblotting. The cytotoxicity induced by knockdown of these antioxidant enzymes was examined by assay of LDH activity. Furthermore, evaluations of lipid peroxidation, cellular levels of reactive oxygen species, cell proliferation, and apoptosis were conducted in cells treated with GPx4 or control siRNA. In oxidative stress study, cells treated with GPx4 or control siRNA were applied with hydrogen peroxide or ferric sulfide, and their cytotoxicity was evaluated by assay of LDH activity. Results Small interfering RNA of catalase, GPx1, GPx4, SOD1, and SOD2 siRNA remarkably inhibited the mRNA and protein expression of each gene. Knockdown of GPx4 and SOD1 but not catalase, GPx1, and SOD2 significantly induced cytotoxicity. Glutathione peroxidase 4 knockdown increased lipid oxidation and reactive oxygen species. The proliferation of GPx4 siRNA-treated cells was reduced compared with control siRNA-treated cells. Moreover, cell death in GPx4 siRNA-treated cells was characterized by positive staining for annexin V. In an oxidation stress study, GPx4 siRNA knockdown enhanced the cytotoxicity induced by hydrogen peroxide or ferric sulfide. Conclusion These results suggest that GPx4 is essential for maintaining oxidative homeostasis and keeping defense against oxidative stress in conjunctival epithelial cells.

In this review, the importance of Japanese research on meibomian gland dysfunction (MGD) is discussed from the perspective of global academic and clinical research on this topic. Many Japanese physicians and researchers have contributed to recent worldwide progress in various fields of MGD research, including pathophysiology, epidemiology, diagnosis, and therapy. In Japan, recent studies in the field of pathophysiology have provided direct evidence for the hypothesis that lipid composition and reactive oxygen species play a crucial role in the development and worsening of MGD. In the field of diagnosis, slit-lamp examination, in vivo confocal microscopy, and meibography have been widely used in studies from Japan. On the basis of the results of these studies, the MGD working group in Japan has proposed new diagnostic criteria for obstructive MGD. According to these criteria, obstructive MGD is considered present when ocular symptoms, anatomic abnormalities, and meibomian gland obstruction are present. In the field of therapy, devices and drugs newly developed in Japan have been shown to enhance the efficacy of lid hygiene and warm compression. Moreover, diquafosol and vitamin D3 have been shown to be effective for MGD. In conclusion, standardization of the diagnosis and treatment of MGD is necessary to enable all patients with MGD to receive appropriate treatment, and specific diagnostic criteria with cutoff values for each parameter are necessary to standardize the diagnosis of MGD.

PURPOSE: To develop a collagen vitrigel (CV) optimized as a corneal endothelial cell (CEC) carrier and create an artificial corneal endothelial graft. METHODS: We first developed a flat-shaped collagen vitrigel for regenerative medicine (CV-RM) using porcine atelocollagen and ultraviolet (UV) irradiation. The optimal UV amount was determined by measuring the CV-RM transparency under various irradiating conditions. The collagen vitrigel for corneal endothelial regenerative treatment (CV-CERT), a transparent porcine atelocollagen with a curved shape, was made using spherically curved molds and UV irradiation. The membrane permeability of the CV-CERT was tested in vitro. The biocompatibility, transparency, and adhesiveness of the CV-CERT were evaluated in rabbit eyes. We also developed a culture technique for distributing human CECs on the curved CV-CERT. RESULTS: The optimal amount of UV irradiation for CV-RM transparency was 2400 mJ/cm(2). Membrane permeability of CV-CERT at day 5 was higher than that of commercially available CV (P = 0.032). The CV-CERT was transparent and biocompatible in rabbit corneas for up to 4 months. The CV-CERT remained attached to the rabbit corneal posterior surface, whereas the flat-shaped CV-RM, differing only in shape from the CV-CERT, dislocated soon after surgery. Human CECs seeded on the CV-CERT using our technique were evenly distributed with a single layer structure and a mean cell density of 2650 ± 100 cells/mm(2). CONCLUSIONS: We developed a transparent and biocompatible porcine-derived atelocollagen vitrigel membrane with a spherical curvature. A transplantable artificial endothelial graft was created by combining cultured human CECs and the CV-CERT.

PURPOSE To evaluate a novel bioengineered corneal endothelial cell sheet that fits the curvature of the posterior corneal surface. METHODS A spherically curved gelatin hydrogel sheet (SCGS) was prepared by the dehydrothermal cross-linking method, and its permeability to water and protein was tested. Monkey corneal endothelial cells (MCECs) were seeded onto these hydrogel sheets, and the cells were examined by immunohistochemistry. Then MCEC-SCGS constructs were transplanted in monkeys with bullous keratopathy to assess the efficacy of the hydrogel sheets as a scaffold. RESULTS The hydrogel sheets showed similar permeability to water and protein as that of atelocollagen and vitrigel sheets. After transplantation, the SCGS did not show wrinkling and adhered tightly to the posterior corneal surface, whereas the flat sheets developed wrinkles that inhibited tight adhesion. Monkey corneal endothelial cells grown on hydrogel sheets expressed anti-zonula occludens-1 (ZO-1), N-cadherin, and sodium, potassium, and adenosine triphosphatase (Na,K-ATPase) along the plasma membrane. In a monkey model of bullous keratopathy, transplanted MCEC-SCGS constructs showed good adhesion to the posterior corneal surface, with subsequent improvement of corneal edema and transparency. CONCLUSIONS A novel MCEC-SCGS construct was effective in a monkey model of bullous keratopathy. The SCGS achieves close adhesion to the posterior corneal surface without wrinkling and may contribute to clinical transplantation of corneal endothelial cell sheets.

PURPOSE To evaluate tear meniscus (TM) changes in external dacryocystorhinostomy (ex-DCR) with quantitative measurement of tear meniscus height (TMH), area (TMA), and volume (TMV) using anterior segment optical coherence tomography (AS-OCT). METHODS Twenty-five eyes from 21 patients (11 males and 10 females) with primary acquired nasolacrimal duct obstruction (PANDO) who received ex-DCR from May 2010 to April 2011 were evaluated prospectively on their TMH, TMA, and TMV changes by AS-OCT. Measurements were performed before surgery (Pre) and 2 weeks (2W), 2 months (2M), and 6 months (6M) after surgery. Data were analyzed using Kruskal-Wallis test, Wilcoxon signed-rank test with Bonferroni adjustment, and Spearman's rank correlation coefficient. RESULTS All patients had a good clinical course, and there were significant differences in the values of each TM parameter before and after surgery (P < 0.0001). The median values of TMH (mm) throughout the observation period were 0.707 (Pre), 0.334 (2W), 0.278 (2M), and 0.277 (6M). The TMA median values (mm(2)) were 0.1097 (Pre), 0.0483 (2W), 0.0255 (2M), and 0.0224 (6M). The TMV median values (mm(3)) were 0.7799 (Pre), 0.1614 (2W), 0.1071 (2M), and 0.1553 (6M). There were significant differences in TMH, TMA, and TMV reduction at each postoperative visit as compared to preoperative values (P < 0.001). In addition, TMH change 6 months after ex-DCR showed a significant positive correlation with age (r = 0.4434, P = 0.0264). CONCLUSIONS The perioperative TM changes in ex-DCR can be evaluated noninvasively and quantitatively by using AS-OCT.

Diagnostic tests of meibomian gland dysfunction (MGD) and of MGD-related disorders are based on the demonstration of abnormal anatomy and physiology of the glands and the detection of specific pathologic events. For this reason, this subcommittee report is divided into two sections. In part I, those aspects of meibomian anatomy and physiology that are relevant to currently available tests are described; a fuller account of the anatomy and physiology is provided in the report of the Anatomy Subcommittee of this workshop. In part II, each test and its performance is described in detail. In part III, the practical application of selected tests is summarized and recommendations for future approaches are made. Additional recommendations and a summary of pertinent literature and concepts are presented in Appendices 1 to 17.

PURPOSE. To analyze outcomes and immunologic features after cultured corneal endothelial cell (CEC) transplantation in a murine model. METHODS. CEC-deprived BALB/c corneas were reconstituted in vitro with an immortalized C3H-CEC cell line and then transplanted orthotopically into recipient BALB/c mice with experimental bullous keratopathy. Graft survival rates, donor-specific delayed hypersensitivity (DTH), and mixed lymphocyte reactions were evaluated in recipient mice after grafting. Fates of CEC transplantation were assessed after adoptive transfer, regrafting, and immunization with C3H splenocytes. RESULTS. Chimeric CEC allografts composed of cultured allogeneic CECs did not provoke rejection reaction, DTH, or mixed-lymphocyte reactions, unlike the high rejection rate that occurred in full-thickness corneal allografts. Adoptive transfer of splenocytes from mice that had accepted chimeric CEC allografts did not increase the graft survival rate after full-thickness corneal transplantation, and the rejection rate of a second full-thickness graft was not improved in these mice, suggestive of no active immunosuppression. Pre-sensitization by subcutaneous injection of splenocytes with the same haplotype as cultured CECs induced systemic DTH to the same allogeneic antigens but did not promote the rejection of chimeric CEC allografts, suggesting that chimeric CEC allografts are ignored by the host immune system. CONCLUSIONS. These findings indicate that immunologic ignorance rather than active immunosuppression is important for the rejection-free acceptance of chimeric CEC allografts. Transplantation of corneal grafts formed with allogeneic CECs could be an ideal treatment strategy to overcome postoperative rejection.

PURPOSE. To establish a culture technique for human corneal epithelial equivalents that do not require fetal bovine serum (FBS), feeder cells, or bovine pituitary extracts and compare this system with conventional culture medium with FBS and mouse 3T3 fibroblasts. METHODS. Human corneal limbal tissue from donor corneas was dissociated on denuded amniotic membranes and then cultured for 3 weeks in feeder-cell‐ and serum-free medium containing epidermal growth factor and B-27. Then, the cell sheet was evaluated by light microscopy, immunohistochemistry, and electron microscopy. The epithelial proliferative capacity was compared between serum- and feeder-cell‐free medium and conventional medium. The cultured cell sheets were transplanted onto the denuded rabbit ocular surface to cover the resected area. RESULTS. A stratified cell sheet expressing cytokeratin-3 and -12 was grown in serum- and feeder-cell‐free medium without unknown growth factors. The epithelial proliferative capacity in feeder-cell‐ and serum-free medium determined by WST-1 and colony-forming efficiency was significantly higher than that in conventional medium. Scanning and transmission electron microscopy showed well-formed stratified epithelium with clear cell boundaries, microvilli, and hemidesmosomal/ desmosomal junctions. The transplanted cell sheets remained transparent without epithelial defects during the follow-up period. CONCLUSIONS. This method using serum- and feeder-cell‐free medium not containing unknown growth factors allows the highly proliferative culture of human corneal epithelium. It avoids exposure of the corneal epithelial equivalent to FBS and animal feeder cells, thus minimizing the risk of contamination by pathogens that could transmit diseases to recipients. (Invest Ophthalmol Vis Sci. 2008;49:2438‐2443) DOI:10.1167/iovs.061448

To investigate the accumulation of new photosensitizers (PSs), dendrimer porphyrin (DP, free DP), and DP encapsulation into polymeric micelles (DP-micelle) and the efficacy of photodynamic therapy (PDT) in an experimental corneal neovascularization model in mice.Corneal neovascularization was induced by suturing 10-0 nylon 1 mm away from the limbal vessel in C57BL6/J mice. To determine the accumulation of free DP and DP-micelle, 10 mg/kg free DP or DP-micelle was administered by intravenous injection 4 days after suture placement. Mice were killed 1, 4, 24, and 168 hours after the injection of PS. Twenty-four hours after the administration of free DP or DP-micelle, mice were treated with a diode laser of 438-nm wavelength at 10 or 50 J/cm(2). Fluorescein angiography was performed before and 7 days after irradiation, and the area of corneal neovascularization was quantified.Free DP and DP-micelle accumulated in the neovascularized area 1 hour to 24 hours after administration. Fluorescence of DP was weaker than that of DP-micelle. Neither DP-micelle nor DP could be detected in normal limbal vasculature. In the PDT experiments using PS, mean residual rates of corneal neovascularization were 10.1% in the mice treated with DP-micelle and 21.6% in the mice treated with free DP at 10 J/cm(2) (P0.01). At 50 J/cm(2), mean residual rates of corneal neovascularization were 10.6% in the mice treated with DP-micelle and 13.7% in the mice treated with free DP (P0.05). Although corneal neovascularization in PDT-treated mice exhibited significant regression compared with the control group, significant energy-related vessel regression with increasing laser energy could not be observed.PDT with DP-micelle and free DP can provide efficacious treatment of corneal neovascularization.

Purpose To characterize the main population of bone marrow-derived cells (BMCs) in human normal subconjunctiva and make a comparison with BMCs in the corneal stroma and epithelium. Methods Normal human donor corneas with attached conjunctiva were examined by fluorescence microscopy after single and double staining for multiple markers. CD68(+) cells were separated from the conjunctival tissues by using magnetic beads, and the expression of toll-like receptor (TLR) 2 and TLR4 was examined. Surface markers of CD68(+) cells were compared with those of BMCs from the corneal stroma and epithelium. Results CD45(+) cells were detected in the substantia propria of the conjunctiva, and approximately 60% of these cells were CD68(+). All the CD68(+) cells expressed HLA-DR and CD14. CD68(+) cells isolated from conjunctival tissues expressed TLR2 and TLR4 on flow cytometry. BMCs in both the corneal stroma and the subconjunctiva expressed scavenger receptor CD163. Macrophage mannose receptor CD206 was expressed by BMCs in the substantia propria of the conjunctiva, but not by BMCs in the corneal stroma or epithelium. Conclusions These findings demonstrated that the main population of BMCs in the substantia propria of normal human conjunctiva is CD68(+)CD14(+)HLA-DR(+) cells. These BMCs express scavenger receptor, macrophage mannose receptor, TLR2, and TLR4 and may play a role in adaptive and innate immune responses in the human ocular surface. These cells are phenotypically different from the CD68(-)CD206(-) monocyte- lineage cells found in the corneal stroma and the CD11c(+)CD68(-)CD163(-)CD206(-) dendritic cells residing in the corneal epithelium.

Purpose To determine the role of macrophage migration inhibitory factor (MIF) in inflammatory corneal neovascularization. Methods Corneal neovascularization was induced by suturing 10-0 nylon 1 mm away from limbal vessel or limbal scraping after 0.15 M NaOH application in BALB/c mice. MIF expression was evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, and immunohistochemistry. To investigate the function of MIF in inflammatory corneal neovascularization, the neovascularized area and number of infiltrating F4/80-positive cells (monocytes/macrophages) were compared between wild-type mice and homozygous MIF-deficient mice. Results MIF mRNA and protein markedly increased in the neovascularized corneas compared with normal corneas by RT-PCR and Western blot analysis, respectively. MIF expression was upregulated immunohistochemically, not only in the corneal epithelium but also in the stromal infiltrating cells of neovascularized corneas. Neovascularized area in corneas of MIF(-/-) mice was significantly small compared with that in wild-type mice on day 7 after corneal suture and on day 14 after limbal scrape, and MIF(-/-) cornea had approximately 30% less neovascularized area than did wild-type cornea in both models. Neovascularized corneas in MIF-deficient mice had significantly fewer monocytes/macrophages than those in wild-type control mice. Conclusions These findings indicate that MIF, abundantly expressed in neovascularized corneas, has an angiogenic role in inflammatory corneal neovascularization and may be a therapeutic target for suppression of corneal neovascularization.

PURPOSE To compare the distribution and self-renewal capacity of rabbit corneal endothelial cell precursors in the central and peripheral regions of the cornea. METHODS The corneal endothelium (CE) and Descemet's membrane of New Zealand White rabbit corneas were divided into a peripheral region (6.0-10.0 mm in diameter) and a central region (6.0 mm in diameter). Then a sphere-forming assay was performed to isolate precursors from the CE of each region. Numbers of primary and secondary sphere colonies and sizes of primary spheres were compared between the central and peripheral regions. RESULTS Primary spheres were isolated from the peripheral and the central regions of the CE. The rate of primary sphere formation in the peripheral region (34.4 +/- 10.4/10,000 cells) was significantly higher than in the central cornea (26.8 +/- 6.6/10,000 cells; P = 0.0042), but there was no significant difference in the size of primary spheres between the two regions. Self-renewal capacity was higher in the peripheral region than in the central region, as evidenced by a significantly higher secondary sphere formation rate for cells from the periphery (39.0 +/- 8.8/10,000 cells) compared with that for cells from the central region (25.4 +/- 4.2/10,000 cells; P = 0.00028). CONCLUSIONS These findings demonstrate that peripheral and central rabbit corneal epithelia contain a significant number of precursors but that the peripheral endothelium contains more precursors and has a stronger self-renewal capacity than the central region.

Purpose To isolate precursors of human corneal endothelial cells (HCECs) in vitro. Methods HCECs were subjected to a sphere-forming assay in which spheres floated in serum-free medium containing growth factors. To promote differentiation, the isolated sphere colonies were plated in dishes coated with poly-L-lysine (PLL)/laminin or fetal bovine endothelium extracellular matrix. Marker expression of neural and mesenchymal cells was examined in the sphere colonies and their progenies by immunocytochemistry and/or reverse transcription-polymerase chain reaction (RT-PCR). Adherent differentiated cells from the sphere colonies were evaluated morphologically and functionally. Results HCECs formed primary and secondary spherical colonies, as shown by sphere-forming assay in vitro. The colonies expressed nestin, beta3-tublin, glial fibrillary acidic protein, and alpha-smooth muscle actin on immunocytochemistry. The progeny, proliferating on extracellular matrix derived from bovine corneal endothelium, but not on PLL/laminin-coated and noncoated dishes, expressed nestin and beta3-tublin. These markers were confirmed by RT-PCR. Adherent differentiated cells from the sphere colonies had an HCEC-like hexagonal shape and satisfactory transport activity that is essential in HCECs. Conclusions These findings indicate that the HCEC contains precursor cells with a propensity to differentiate into HCECs and that these cells can also produce neuronal and mesenchymal cell proteins.

PURPOSE. To evaluate the function of cultured human corneal endothelial cells (HCECs) in vivo and the feasibility of HCEC transplantation with a collagen sheet as the substitute carrier of HCECs. METHODS. Adult human donor cornea derived from cultured HCECs was labeled with the fluorescent tracker DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) and seeded on a collagen sheet. The pump function of the HCEC sheet was evaluated by measurement of the potential difference and short-circuit current. A 6-mm sclerocorneal incision and Descemetorhexis were performed on rabbit eyes. The HCECs on a collagen sheet was brought into the anterior chamber and fixed to the posterior stroma (HCEC group). Rabbit corneas with collagen sheet transplantation after Descemetorhexis (collagen group) and with only Descemetorhexis (no-transplantation group) were the control. Each group, observed for 28 days after surgery, underwent histologic and fluorescence microscopic examinations. RESULTS. Pump function parameters of the HCEC sheets were 76% to 95% of those of human donor corneas. Mean corneal thickness in the HCEC group was significantly less than in the collagen and no-transplantation groups 1, 3, 7, 14, 21, and 28 days (P < 0.05) after surgery. DiI-labeled cells were spread over the rear corneal surface in the HCEC group. Marked stromal edema was present in the collagen and no-transplantation groups with hematoxylin-eosin staining, hut none in the HCEC group with collagen sheets hearing monolayer cells. CONCLUSIONS. The findings indicate that cultured HCECs transplanted from adult human donor cornea by means of a collagen sheet can retain their function of corneal dehydration in a rabbit model and suggest the feasibility of transplantation for CEC dysfunction using cultured HCECs with a collagen sheet.