Altered expression of aquaporins in bullous keratopathy and Fuchs' dystrophy corneas.

Corneas with edema-related diseases lose transparency, which causes significant vision loss. This study analyzed seven aquaporins (AQPs) in normal corneas, pseudophakic/aphakic bullous keratopathy (PBK/ABK) corneas, Fuchs' dystrophy corneas, keratoconus corneas, post-cataract surgery (PCS) corneas, and normal organ-cultured corneas. RNA levels for AQP1, AQP4, and beta2-microglobulin were measured by RT-PCR. AQP1 antibody localized to stromal cells of all corneas. PBK/ABK and Fuchs' dystrophy corneas had decreased endothelial cell staining compared with normal. AQP1 mRNA was found in whole corneas and cultured stromal fibroblasts but not in isolated epithelial cells. AQP3 staining was found in basal epithelial cells of the normal, Fuchs' dystrophy, and keratoconus corneas but throughout the entire epithelium of PBK/ABK corneas. AQP4 antibody localized to endothelial cells of all corneas and in stromal cells of PBK/ABK corneas. AQP4 mRNA was identified in whole human corneas. AQP5 was found in epithelial cells of all corneas. AQP0, AQP2, and AQP9 were not found in any corneas. Normal AQP distributions were found in PCS and organ-cultured corneas, although they showed signs of swelling. Our study demonstrates that AQP abnormalities are found in PBK/ABK corneas (decreased AQP1, increased AQP3 and AQP4) and Fuchs' dystrophy corneas (decreased AQP1). Although both have vision-disrupting corneal edema, the mechanisms of fluid accumulation may be different in each disease.     

The effect of cryoprotection and cryodamage on corneal rehydration and endothelial structure

Corneal rehydration rate and endothelial ultrastructure were compared during postthaw perfusion of cryopreserved corneas and corneas that had been frozen and thawed without the benefit of cryoprotective solutions. The postthaw swelling rate of cryopreserved rabbit corneas was 171 μm/hr compared to 333 μm/hr in the cryodamaged corneas. Most of the endothelial cells were intact in the cryopreserved corneas, but the intercellular spaces were enlarged. In contrast, all of the endothelial cells in the cryodamaged corneas were disrupted and Descemet's membrane was only partially covered by the remaining cell fragments.     

Quantitative changes and ultrastructural alterations of the cornea in response to ultraviolet light. II. Effects of amphibia; elucidation of desmosomal structure and basement membrane synthesis.

The effects of far ultraviolet light irradiation upon an amphibian cornea were studied to compare the effects observed both quantitatively and ultrastructurally with data obtained after UV irradiation of mammalian corneas. The ultimate goal of this series of investigations is the elucidation of the alterations and the regeneration mechanisms, which might reflect existing morphological diversities among the species, observed in vertebrate corneas following exposure to UV light. It was found that while the epithelial cells undergo oedema after low dose exposures and are gradually damaged after high doses of UV light, 2-4 days leter a new epithelium has been formed. Intercellular permeability is increased by low dose exposure as was detected by the penetration of Ruthenium Red into the intercellular clefts. Under these conditions desmosomal structure revealed a 21-laminar configuration. The basement membrane of the amphibian, unlike that of the mammal, does not dissolve away upon exposure but shows localized disruptions which are thought to accommodate the passage of leucocytes from stroma to epithelium. That a new basement membrane is subsequently formed is evident by the existence of extracellular and intracellular secretion granules. In comparison to irradiated rabbit corneas, this stroma remains remarkably at the same thickness following a high dose exposure although a noticeable disorganization of collagen arrangement is apparent. Finally, as in the case of the rabbit corneas, a secondary degeneration of endothelium was observed 4 days after a moderate dose exposure.     

Regulation of corneal collagenase production: stimulation of serially passaged stromal cells by blood mononuclear cells.

After three or more passages, stromal cell outgrowths from explants of normal rabbit corneas and from corneas previously burned with alkali did not produce collagenase until they were stimulated by the addition of rabbit blood-derived mononuclear cells or media conditioned by them. Normal stromal cells required stimulation by lymphocytes or their products from alkali-burned animals, whereas those from alkali-burned corneas were stimulated by lymphocytes from either normal or alkali-burned rabbits.     

Cell adhesion molecules in stromal corneal dystrophies

The aim of the present study was to investigate the expression pattern of different cell adhesion molecules in corneal stromal dystrophies. Fifteen corneal buttons from patients diagnosed with three different types of stromal corneal dystrophies and healthy corneas were investigated. Paraffin embedded sections were stained immunohistochemically with monoclonal antibodies against human intercellular adhesion molecule-1 (ICAM-1), endothelial selectin (E-selectin) and endothelial cadherin (E-cadherin) using the avidin-biotin-peroxidase-complex technique. The sections were compared to normal eye bank controls. In corneas from granular dystrophy patients ICAM-1 was expressed focally in epithelial cells and in keratocytes, and expressed diffusely in endothelial cells. In corneas from macular dystrophy patients diffuse epithelial staining was observed and the stromal and endothelial expression was found to be similar to that of granular dystrophy. In lattice dystrophy, only the epithelial cells and endothelium were intensively positive for ICAM-1. E-selectin was not present on any layer of the corneal specimens. E-cadherin was observed only in the epithelium of all three types of corneal dystrophies. Normal corneas did not express any of the investigated adhesion molecules. We found different expression patterns of adhesion molecules in corneas from stromal dystrophies. Our results suggest that adhesion molecules may be involved in the pathogenesis of corneal stromal dystrophies.     

Comparison of three methods for human corneal cryopreservation that utilize dimethyl sulfoxide.

We compared endothelial cell survival in human corneas after cryopreservation by three methods that utilize dimethyl sulfoxide. Twenty-eight human cadaver corneas were cryopreserved by one of three methods, stored briefly over liquid nitrogen, thawed, cultured at 37 degrees C for 3 days, and fixed for scanning electron microscopy. Seventeen control corneas underwent identical cryoprotectant immersion and culture protocols but were not frozen. Endothelial photographs taken after 1 and 3 days of culture were analyzed. Endothelial cell losses in cryopreserved corneas by Methods 1, 2, and 3, respectively, were 36, 22, and 10% after 1 day of culture and 57, 36, and 27% after 3 days of culture. Cryopreservation by Method 3 had less cell loss than Methods 1 or 2 (P<0.02) but greater cell loss than the control corneas for Method 3 (P<0.001). No loss of cells occurred in the control corneas for Methods 1 and 3 but substantial cell loss (26%) occurred in the control corneas for Method 2. Polymegethism and pleomorphism of the endothelial cells were seen in the corneas that lost cells. The endothelial cell loss of 10% seen after 1 day of culture in human corneas cryopreserved by Method 3 is similar to the loss that occurs during organ culture storage as currently used clinically and therefore would be acceptable for clinical use. After 3 days of culture, however, the cell loss had increased significantly to 27%. This additional decrease in cell number that occurs in culture may represent latent cryodamage and must be understood and overcome in vivo before the technique can be used clinically.     

Evaluation of acute corneal damage induced by 222-nm and 254-nm ultraviolet light in Sprague–Dawley rats

Two hundred twenty-two nanometres ultraviolet (UV) light produced by a krypton–chlorine excimer lamp is harmful to bacterial cells but not skin. However, the effects of 222-nm UV light exposure to the eye are not fully known. We evaluated acute corneal damage induced by 222- and 254-nm UV light in albino rats. Under deep anaesthesia, 6-week-old Sprague–Dawley albino rats were exposed to UV light. The exposure levels of corneal radiation were 30, 150, and 600?mJ/cm². Epithelial defects were detected by staining with fluorescein. Superficial punctate keratitis developed in corneas exposed to more than 150?mJ/cm² of UV light, and erosion was observed in corneas exposed to 600?mJ/cm² of UV light. Haematoxylin and eosin staining also showed corneal epithelial defects in eyes exposed to 254-nm UV light. However, no damage developed in corneas exposed to 222-nm UV light. Cyclobutane pyrimidine dimer-positive cells were observed only in normal corneas and those exposed to 254-nm UV light. Although some epithelial cells were stained weakly in normal corneas, squamous epithelial cells were stained moderately, and the epithelial layer that was detached from the cornea exposed to 600?mJ/cm² of light was stained intensely in corneas exposed to 254-nm UV light. In the current study, no corneal damage was induced by 222-nm UV light, which suggested that 222-nm UV light may not harm rat eyes within the energy range and may be useful for sterilising or preventing infection in the future.     

Keratocyte injury in human corneas cryopreserved under standard conditions

This study was conducted to characterize ultrastructural damage to human corneas cryopreserved by a standard protocol. The materials used were seven human corneas that were unsuitable for transplantation due to the presence of positive bacteriological cultures; they were cryopreserved according the standard procedure. After freezing and thawing, samples were obtained for scanning and transmission electron microscopy studies. Marked damage was observed in keratocytes with signs of apoptotic cellular injury. However our observations have shown that apoptosis contribute less significantly than necrosis to cellular death in keratocytes of human corneas and although the control of apoptosis is clearly desirable, in order to improve the success of cryopreserved corneas for transplant, we need to continue our investigation to reduce the effects of the necrotic process. This revised version was published online in July 2006 with corrections to the Cover Date.     

Preparation of pre-cut corneas from fresh donated whole globes for Descemet’s stripping automated keratoplasty: 3-year results at the Central Eye Bank of Iran

To describe the technique and the results of the preparation of pre-cut corneas for Descemet’s stripping automated endothelial keratoplasty (DSAEK) during a 3-year period at the Central Eye Bank of Iran (CEBI). The method of preparation of pre-cut corneas from donated whole globes at the CEBI is described and the frequency and percentage of pre-cut corneas prepared for DSAEK, between April 2009 and March 2012, are specified. Moreover, post-operative reports are reviewed for any complaints about using pre-cut tissues for DSAEK. Out of the 1,518 donated whole globes appropriate for DSAEK, 1,478 (97.4 %) pre-cut corneas were successfully prepared. The method of preparation failed in 40 (2.6 %) cases. Based on the eye bank post-operative reports, thickness of pre-cut tissues for DSAEK was deemed unacceptable in only 6 (0.4 %) cases prior to surgery; five of these were too thick and one was too thin. Preparation of pre-cut corneas, for DSAEK from donated whole globes, in the CEBI is a safe and easy method, with very good preservation of endothelial cells after the preparation of the pre-cut corneas and reduced risks from corneal manipulation.     

Expansion and cryopreservation of porcine and human corneal endothelial cells

Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me₂SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me₂SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine.     

Distribution of TGF‐β isoforms and signaling intermediates in corneal fibrotic wound repair

In this study, temporal and spatial distribution of three TGF‐β isoforms and their downstream signaling pathways including pSmad2 and p38MAPK were examined during fibrotic wound repair. In normal chick corneas, TGF‐β1, ‐2, and ‐3 were weakly detected in Bowman's layer (BL). In healing corneas, TGF‐β1 was primarily deposited in the fibrin clot and the unwounded BL. TGF‐β2 was highly expressed in healing epithelial and endothelial cells, and numerous active fibroblasts/myofibroblasts. TGF‐β3 was mainly detected in the unwound region of basal epithelial cells. α‐Smooth muscle actin (α‐SMA) was initially appeared in the posterior region of repairing stroma at day 3, and was detected in the entire healing stroma by day 7. Notably, α‐SMA was absent in the central region of healing stroma by day 14, and its staining pattern was similar to those of TGF‐β2 and p38MAPK. By contrast, pSmad2 was mainly detected in the fibroblasts. In normal cornea, laminin was mainly detected in both epithelial basement membrane (BM) and Descemet's membrane (DM). By contrast to reconstitution of the BM in the wound region, the DM was not repaired although endothelial layer was regenerated, indicating that high levels of TGF‐β2 were released into the posterior region of healing stroma on day 14. High levels of α‐SMA staining, shown in cultured repair stromal cells from healing corneas on day 14 and in TGF‐β2 treated normal stromal cells, were significantly reduced by p38MAPK inhibition. Collectively, this study suggests that TGF‐β2‐mediated myofibroblast transformation is mediated, at least partly, by the p38MAPK pathway in vivo. J. Cell. Biochem. 108: 476–488, 2009. © 2009 Wiley‐Liss, Inc.     

Heterogeneity in keratoconus: possible biochemical basis

Total protein and collagen content in normal and keratoconus corneas were determined. The protein content (expressed as a function of dry weight) in all keratoconus corneal samples was lower than that found in normal corneas. However, among the 11 keratoconus corneas examined, only 7 (group A) had the same hydroxyproline content (expressed as a function of dry weight) as normal corneas; 4 others (group B) showed significantly less. In tissue culture, four strains derived from keratoconus stroma (group I) produced total protein at the same rate as cells from normal controls. Four other strains (group II), however, had a decreased rate of protein synthesis. The amount of collagenous protein synthesized per microgram DNA by group I strains was similar to that found in normal cultures, whereas it was significantly reduced in group II cultures. We suggest that group I strains represent group A corneas. Group II strains, with a reduced level of both protein and collagen synthesis, may represent group B corneas. The defect in this group appears to be decreased total synthetic activity of corneal cells. The variation in our results suggests that keratoconus is a heterogeneous disease. The heterogeneity may explain the contradictory data that exist in the literature.     

Impairment of thrombospondin-1 expression during epithelial wound healing in corneas of vitamin A-deficient mice

The purpose of this study is to investigate the expression of thrombospondin-1 (TSP-1), a multifunctional extracellular matrix protein, during re-epithelialization in wounded corneas of vitamin A-deficient mice. Epithelial defects were created in the corneas of normal and Vitamin A-deficient mice with a microgrinder. Wounded corneas were stained with fluorescein and photographed for evaluation of re-epithelialization. Histological examination and immunohistochemical analysis of TSP-1 expression were also performed on the specimens from wounded corneas. In vitamin A-deficient mice, re-epithelialization of the wounded corneal epithelium was significantly delayed compared with that in normal mice. TSP-1 was detectable neither in the unwounded corneal epithelium of normal mice nor in that of vitamin A-deficient mice. In normal mice, linear staining of TSP-1 was observed on the wounded corneal surface and stroma at 30 min and 8 h to 16 h, respectively, after abrasion, and this TSP-1 expression disappeared at 36 to 48 h, when re-epithelialization was completed. In contrast, no TSP-1 staining was observed in the wounded corneas of vitamin A-deficient mice, except for the endothelial cells, throughout the wound healing process. Histological examination revealed a progressive increase in polymorphonuclear neutrophil infiltration in the stroma of the corneas of vitamin A-deficient mice during the healing process. These findings suggest that vitamin A may modulate the expression of TSP-1 in the corneas to accelerate the re-epithelialization of wounded corneas.     

Optimization of a Method for the Cryopreservation of Rabbit Corneas: Attempted Application to Human Corneas

The purpose of the present study was to set up and test a cryopreservation method for long-term storage of human corneas. Therefore the freezing solution was optimized in 264 rabbit corneas by testing the type of cryoprotectant, its concentration, addition and dilution pattern and exposure temperature. Then rabbit corneas were frozen in the optimum solution at different cooling rates and thawed in a water bath at different temperatures. Eight human corneas were cryopreserved with the method showing optimum results in rabbit corneas and four additional corneas were used as controls. Endothelial viability was assessed after each step by vital staining and scanning electron microscopy. Best results after exposure of rabbit corneas to the freezing solution were achieved when using a 10% cryoprotectant concentration, with direct addition/dilution and exposure at room temperature (3512 ±300 viable cellsmm² when using dimethylsulfoxide; 3403 ± 245 viable cellsmm² when using 1,2-propanediol). Cryopreserved rabbit corneas had the highest endothelial cell survival when frozen at 1°C/min and thawed at 37°C (2003 ± 372 viable cells/mm² when using dimethylsulfoxide and 1357 ± 667 viable cells/mm² when using 1,2-propanediol). Cryopreserved human corneas had 753 ± 542 viable cells/mm² when using dimethylsulfoxide and 56 ± 56 viable cells/mm² when using 1,2-propanediol. We can conclude that the method developed is easy to handle and shows optimum results in rabbit corneas, with an endothelial cell survival that is consistent with transplant acceptability criteria. The results obtained in human corneas are below prediction and are still unsatisfactory for successful use in eye banking.     

Oligoadenylate synthetase/protein kinase R pathways and alphabeta TCR+ T cells are required for adenovirus vector: IFN-gamma inhibition of herpes simplex virus-1 in cornea

An adenoviral (Ad) vector containing the murine IFN-gamma transgene (Ad:IFN-gamma) was evaluated for its capacity to inhibit HSV-1. To measure effectiveness, viral titers were analyzed in cornea and trigeminal ganglia (TG) during acute ocular HSV-1 infection. Ad:IFN-gamma potently suppressed HSV-1 replication in a dose-dependent fashion, requiring IFN-gamma receptor. Moreover, Ad:IFN-gamma was effective when delivered -72 and -24 h before infection as well as 24 h postinfection. Associated with antiviral opposition, TG from Ad:IFN-gamma-transduced mice harbored fewer T cells. Also related to T cell involvement, Ad:IFN-gamma was effective but attenuated in TG from alphabeta TCR-deficient mice. In corneas, alphabeta TCR(+) T cells were obligatory for protection against viral multiplication. Type I IFN involvement amid antiviral efficacy of Ad:IFN-gamma was further investigated because types I and II IFN pathways have synergistic anti-HSV-1 activity. Ad:IFN-gamma inhibited viral reproduction in corneas and TG from alphabeta IFNR-deficient (CD118(-/-)) mice, although viral titers were 2- to 3-fold higher in cornea and TG compared with wild-type mice. The absence of IFN-stimulated antiviral proteins, 2'-5' oligoadenylate synthetase/RNase L, and dsRNA-dependent protein kinase R completely eliminated the antiviral effectiveness of Ad:IFN-gamma. Collectively, the results demonstrate the following: 1) nonexistence of type I IFN receptor does not abolish defense of Ad:IFN-gamma against HSV-1; 2) antiviral pathways oligoadenylate synthetase-RNase L and protein kinase R are mandatory; and 3) alphabeta TCR(+) T cells are compulsory for Ad:IFN-gamma effectiveness against HSV-1 in cornea but not in TG.     

Maintenance and Distribution of Epithelial Stem/Progenitor Cells after Corneal Reconstruction Using Oral Mucosal Epithelial Cell Sheets

We assessed the maintenance and distribution of epithelial stem/progenitor cells after corneal reconstruction using tissue-engineered oral mucosal cell sheets in a rat model. Oral mucosal biopsy specimens were excised from green fluorescent protein (GFP) rats and enzymatically treated with Dispase II. These cells were cultured on inserts with mitomycin C-treated NIH/3T3 cells, and the resulting cell sheets were harvested. These tissue-engineered cell sheets from GFP rats were transplanted onto the eyes of a nude rat limbal stem cell deficiency model. Eight weeks after surgery, ocular surfaces were completely covered by the epithelium with GFP-positive cells. Transplanted corneas expressed p63 in the basal layers and K14 in all epithelial layers. Epithelial cells harvested from the central and peripheral areas of reconstructed corneas were isolated for a colony-forming assay, which showed that the colony-forming efficiency of the peripheral epithelial cells was significantly higher than that of the central epithelial cells 8 weeks after corneal reconstruction. Thus, in this rat model, the peripheral cornea could maintain more stem/progenitor cells than the central cornea after corneal reconstruction using oral mucosal epithelial cell sheets.     

P53, CD95, cathepsin and survivin pathways in Fuchs' dystrophy and pseudophakic bullous keratopathy corneas

Our purpose was to elucidate the pathways of apoptosis of corneas with Fuchs' dystrophy and pseudophakic bullous keratopathy. Sixteen corneal buttons (14 patients, median age 73 years) with Fuchs' dystrophy, 13 with pseudophakic bullous keratopathy (PBK) (13 patients, median age 69 years) and 8 buttons (8 patients, median age 59 years) from enucleated eyes with chorioideal melanoma (controls) were analysed histologically. Immunohistochemical analysis was performed to investigate the expression of p21, p27, p63, survivin, CD95, cathepsin, bax, bcl-2 and Ki67. Positive immunohistochemical reactions were detected in epithelial cells of the corneas, but keratocytes and endothelial cells were not positive in any of the groups or stainings. The number of p27 and survivin positive epithelial cells was significantly lower (p=0.048 and 0.041) and the number of cathepsin positive epithelial cells was significantly higher (p=0.004) in Fuchs' dystrophy corneas compared to controls. In pseudophakic bullous keratopathy, p21 and p27 positive epithelial cells were present in a significantly lower (p=0.02 and 0.005) number than in controls. We conclude that genetically programmed cell death is related to the p27, cathepsin and survivin pathways in Fuchs' dystrophy and to the p21 and p27 pathways in pseudophakic bullous keratopathy.     

Analysis of corneal development with monoclonal antibodies. I. Differentiation in isolated corneas

Monoclonal antibodies highly selective for developmentally regulated antigens present in the cornea (Zak and Linsenmayer, Dev. Biol. 99, 373-381, 1983) have been used to immunohistochemically evaluate differentiation in intact chick corneas cultured on the chorioallantoic membrane (CAM) of host embryos. One antibody is directed against the epithelial cell layer and the other is against the corneal stromal matrix. It has been established that both antigens recognized by the antibodies are expressed de novo in young explanted corneas and that the stromal matrix antigen is a product of the corneal fibroblasts. Thus expression of the antigens can be used as criteria for overt differentiation of the respective cell types. The antibodies have been employed to assess when the corneal epithelial and stromal cells become capable of autonomous differentiation within isolated corneas. To accomplish this, corneas of various ages were explanted with and without adjacent pericorneal tissues. The results indicate that, under the culture conditions employed, corneal stromal differentiation is dependent on the presence of the lens until stage 28 (51/2-6 days of development), which is the time when invasion of the stroma by pericorneal mesenchymal cells is initiated. After stage 28, the stromal matrix antigen was expressed by isolated corneas irrespective of the presence of the lens. Possibly the lens acts by maintaining the integrity of the corneal endothelial monolayer and thus promoting normal migration of pericorneal mesenchymal cells into the primary corneal stroma, where they undergo differentiation. Conversely, differentiation of the corneal epithelium was independent of any pericorneal structure from the earliest stage examined (41/2-5 days of development). It was even independent of overt stromal differentiation, thus suggesting an early and strong determination for this tissue.     

Atomic force microscopy analysis of normal and photoablated porcine corneas

We showed the capabilities and accuracy of atomic force microscopy (AFM) techniques for imaging and analyzing the corneal epithelium and the photoablated corneal stroma. Eight normal porcine corneas, half of which were ablated using a scanning-spot excimer laser, were examined. All the corneas were imaged in balanced salt solution after fixation in glutaraldehyde. In the normal untreated corneas we observed the epithelial surface showing the typical polygonal cells and presenting numerous microprojections. The superficial epithelial cells were classified in three types as a result of the anterior-surface roughness measurements. AFM images of the photoablated corneal specimens showed undulations and granule-like features on the ablated stromal surface, specific to 193-nm ArF laser irradiation. Nevertheless, the quantitative analysis confirmed the precision of excimer laser surgery in removing sub-micrometric amounts of tissue. AFM showed to be a high-resolved imaging tool for the scanning of both native as well as photoablated corneal specimens. Also, this technique permits precise topographic analysis of the corneal plane, in the nanometric scale, of which smoothness is an important physical characteristic and necessary to achieve an optimal optical quality of the eye.