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The cornea is a fundamental ocular tissue for the sense of sight. Thanks to it, the refraction of two-thirds of light manages to participate in the visual process and protect against mechanical damage. Because it is transparent, avascular, and innervated, the cornea comprises five main layers: Epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. Each layer plays a key role in the functionality and maintenance of ocular tissue, providing unique ultrastructural and biomechanical properties. Bullous Keratopathy (BK) is an endothelial dysfunction that leads to corneal edema, loss of visual acuity, epithelial blisters, and severe pain, among other symptoms. The corneal layers are subject to changes in their biophysical properties promoted by Keratopathy. In this context, the Atomic Force Microscopy (AFM) technique in air was used to investigate the anterior epithelial surface and the posterior endothelial surface, healthy and with BK, using a triangular silicone tip with a nominal spring constant of 0.4 N/m. Six human corneas (n = 6) samples were used for each analyzed group. Roughness data, calculated by third-order polynomial adjustment, adhesion, and Young's modulus, were obtained to serve as a comparison and identification of morphological and biomechanical changes possibly associated with the pathology, such as craters and in the epithelial layer and exposure of a fibrotic layer due to loss of the endothelial cell wall. Endothelial cell membrane area and volume data were calculated, obtaining a relevant comparison between the control and patient. Such results may provide new data on the physical properties of the ocular tissue to understand the physiology of the cornea when it has pathology., Competing Interests: Declaration of competing interest All authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Corneal transplantation is the only way to treat serious corneal diseases caused by corneal endothelial dysfunction. However, the shortage of donor corneal tissues and human corneal endothelial cells (HCECs) remains a worldwide challenge. We cultivated HCECs by the use of a conditioned medium from orbital adipose-derived stem cells (OASC-CM) in vitro. Then the HCECs were used to treat animal corneal endothelial dysfunction models via cell transplantation. The purpose of this study was to conduct a long-term observation and evaluation after cell transplantation., Methods: Orbital adipose-derived stem cells (OASCs) were isolated to prepare the conditioned medium (CM). HCECs were cultivated and expanded by the usage of the CM (CM-HCECs). Then, related corneal endothelial cell (CEC) markers were analyzed by immunofluorescence. The cell proliferation ability was also tested. CM-HCECs were then transplanted into monkey corneal endothelial dysfunction models by injection. We carried out a 24-month postoperative preclinical observation and verified the long-term effect by histological examination and transcriptome sequencing., Results: CM-HCECs strongly expressed CEC-related markers and maintained polygonal cell morphology even after 10 passages. At 24 months after cell transplantation, there was a CEC density of more than 2400 cells per square millimeter (range, 2408-2685) in the experimental group. A corneal thickness (CT) of less than 550 μm (range, 490-510) was attained. Gene sequencing showed that the gene expression pattern of CM-HCECs was similar to that of transplanted cells and HCECs., Conclusions: Transplantation of CM-HCECs into monkey corneal endothelial dysfunction models resulted in a transparent cornea after 24 months. This research provided a promising prospect of cell-based therapy for corneal endothelial diseases., (© 2022. The Author(s).)

Human corneal endothelial cells (CECs) have limited ability to regenerate in vivo. Oxidative stress has been proposed as one potential reason. Understanding the mechanism of oxidative stress-induced CEC dysfunction might provide novel targets for improving CEC regenerative capacity, and help develop non-surgical therapeutic strategies for CEC dysfunction. Long non-coding RNAs (lncRNAs) are non-coding transcripts with multiple biological functions. The roles of lncRNAs in ocular cells under oxidative stress have been widely studied, such as lens epithelial cells, trabecular meshwork cells, and retinal ganglion cells. In the current study, we established oxidative stress-induced CEC dysfunction model in vitro. By RNA sequencing technology, we identified 824 differentially expressed lncRNAs in CEC dysfunction group, including 667 upregulated lncRNAs and 157 downregulated lncRNAs. We finally demonstrated that CEC functions under oxidative stress, including cellular proliferation, apoptosis, and anti-oxidative stress ability, could be regulated by different lncRNAs, including lncRNA-Z93241.1, lncRNA-XLOC_000818, and lncRNA-AC007952.4. Targeting these lncRNAs might be useful to further elucidate the pathology of CEC dysfunction and develop novel therapeutic strategy., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Corneal disease affects 12.5 million individuals worldwide, with 2 million new cases each year. The standard treatment consists of a corneal transplantation from a human donor; however, the worldwide demand significantly exceeds the available supply. Lamellar endothelial keratoplasty, the replacement of only the endothelial layer of the cornea, can partially solve the problem. Progressive efforts have succeeded in expanding hCECs; however, the ability to expand hCECs is still limited, and new sources of CECs are being sought. Crucial advances have been achieved by the directed differentiation of embryonic or induced pluripotent stem cells, but these cells have disadvantages, such as the use of oncogenes, and are still difficult to establish. We aimed to transfer such knowledge to obtain hCECs from adipose tissue-derived adult mesenchymal stem cells (ADSC) by modifying four previously published procedures. We present several protocols capable of the directed differentiation of human ADSCs to hCECs. In our hands, the protocol by Ali et al. was the best adapted to such differentiation in terms of efficiency, time, and financial cost; however, the protocol by Wagoner et al. was the best for CEC marker expression. Our results broaden the type of cells of autologous extraocular origin that could be employed in the clinical setting for corneal endothelial deficiency.

The shortage of human donor corneas has raised important concerns about engineering of corneal endothelial cells (CECs) for clinical use. However, due to the limited proliferative capacity of human CECs, driving them into proliferation and regeneration may be difficult. Unlike human CECs, rabbit CECs have a marked proliferative capacity. To clarify the potential reason for this difference, we analysed the proteomes of four human corneal endothelium samples and four rabbit corneal endothelium samples with quantitative label-free proteomics and downstream analysis. We discovered that vitamin and selenocompound metabolism and some signaling pathways such as NF-kappa B signaling pathway differed between the samples. Moreover, TGFβ, PITX2 and keratocan were distinctively expressed in rabbit samples, which might be associated with active proliferation in rabbit CECs. This study illustrates the proteomic differences between human and rabbit CECs and might promote CEC engineering strategies., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: Corneal endothelial dysfunction leads to corneal edema, pain, and vision loss. Adequate animal models are needed to study the safety and efficacy of novel cell therapies as an alternative to corneal transplantation., Methods: Primary human corneal endothelial cells (HCECs) were isolated from cadaveric donor corneas, expanded in vitro, transduced to express green fluorescent protein (GFP), loaded with superparamagnetic nanoparticles, and injected into the anterior chamber of adult rabbits immediately after endothelial cell or Descemet's membrane stripping. The same volume of balanced salt solution plus (BSS+) was injected in control eyes. We compared different models for inducing corneal edema in rabbits, and examined the ability of transplanted HCECs to reduce corneal edema over time by measuring central corneal thickness and tracking corneal clarity. GFP-positive donor cells were tracked in vivo using optical coherence tomography (OCT) fluorescence angiography module, and the transplanted cells were confirmed by human nuclei immunostaining., Results: Magnetic HCECs integrated onto the recipient corneas with intact Descemet's membrane, and donor identity was confirmed by GFP expression and immunostaining for human nuclei marker. Donor HCECs formed a monolayer on the posterior corneal surface and expressed HCEC functional markers of tight junction formation. No GFP-positive cells were observed in the trabecular meshwork or on the iris, and intraocular pressure remained stable through the length of the study., Conclusions: Our results demonstrate magnetic cell-based therapy efficiently delivers HCECs to restore corneal transparency without detectable toxicity or adverse effect on intraocular pressure. Magnetic delivery of HCECs may enhance corneal function and should be explored further for human therapies.

There are no effective treatments for corneal endothelial diseases, except for corneal transplantation, as human corneal endothelial cells (hCECs) do not regenerate. The regeneration of hCECs could be induced through regulation of the expression of specific genes. In this study, we investigated whether the overexpression of sex-determining region Y-box 2 (SOX2) can regenerate hCECs in vivo and in vitro. SOX2 was activated using the clustered regularly interspaced short palindromic repeats (CRISPR)/deactivated CRISPR-associated protein 9 (dCas9) activation system. Genes were transfected into the corneal endothelium of Sprague-Dawley rats. Central corneal thickness and opacity were measured, and alizarin red S staining was performed. Corneal opacity and central corneal thickness were reduced in the SOX2 group compared with the control group. The density of CECs was higher in the SOX2 group compared with the control group. Additionally, hCECs were cultured and analyzed after overexpressing SOX2. Cell viability, proliferation rate, and the number of cells in S-phase were increased after SOX2 overexpression (p < .05). Cyclin-dependent kinase 1 and cyclin D1 were found to be overexpressed (p < .05). WNT signaling was repressed, and the AKT pathway was activated by SOX2 overexpression. Mitochondrial oxidative stress and energy production were increased by SOX2 overexpression (p < .05). In conclusion, SOX2 activation promotes wound healing and regeneration in CECs. SOX2 activation using the CRISPR/dCas9 system may thus be useful for the treatment of hCEC diseases. Stem Cells 2018;36:1851-12., (© 2018 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Purpose: To investigate functional and anatomical results up to 12 months after Descemet's membrane endothelial keratoplasty (DMEK) for Fuchs' endothelial dystrophy (FED) versus bullous keratopathy (BK) in consideration of morphologic characteristics on host's endothelium-Descemet's membranes (EDM)., Methods: In a prospective consecutive case series, 119 eyes underwent a primary DMEK for FED or BK. Intraoperatively obtained EDM were investigated immunohistologically. Clinical and morphological parameters were compared between FED and BK., Results: Overall, the 12-months best-corrected visual acuity (BCVA) was 0.14 logMAR, and 0.10 logMAR in eyes without vision-limiting comorbidities; thereby no differences were revealed between eyes with FED and BK up to 12 months postoperative (p = 0.186 and p = 0.095, respectively). Correspondingly, the mean central corneal thickness (CCT) measured 520 vs. 529 µm and the mean endothelial cell density (ECD) was 1743 vs. 1457 cells/mm 2 for FED and BK, 12 months postoperative. Regarding CCT and ECD, no differences were observed between the groups (p = 0.181 and p = 0.112, respectively). The overall detachment rate was 40% (48/119). Comparing FED and BK the detachment rates did not differ, which were 41% vs. 39% and 43% vs. 35%, in pseudophakic eyes (p = 0.554 and p = 0.601, respectively). Yet, the distribution of recurring graft detachments differed between FED and BK; secondary re-detachments were more frequent in the FED group (7 cases). Regarding histologic investigations, a lower ECD was found in specimens with BK, no differences were revealed for EDM and anterior banded layer thicknesses. Immunohistologically, differences in the distribution of fibronectin and cytokeratin were observed. A rarification of matrix proteins was found in EDM complexes with FED., Conclusions: DMEK produces similar results for FED and BK. However, the postoperative course may differ with regard to the recurrence of secondary graft detachments that may be associated by histopathologic particularities.

Corneal endothelial cells (CECs) are essential for maintaining corneal stromal hydration and ensuring its transparency, which is necessary for normal vision. Dysfunction of CECs leads to stromal decompensation, loss of transparency and corneal blindness. Corneal endothelium has low proliferative potential compared to surface epithelial cells leading to poor regeneration of CEC following injury. Additionally, the tissue exhibits age related decline in endothelial cell density with re-organisation of the cell layer, but no regeneration. The mechanisms which control proliferation and differentiation of neural crest derived CEC progenitors are yet to be clearly elucidated. Prdm (Positive regulatory domain) family of transcriptional regulators and chromatin modifiers are important for driving differentiation of a variety of cellular types. Many Prdm proteins are expressed in specific precursor cell populations and are necessary for their progression to a fully differentiated phenotype. In the present work, we sought to identify members of the Prdm gene family which are specifically expressed in human (h) CECs with a view to begin addressing their potential roles in CEC biology, focussing especially on Prdm 4 and 5 genes. By performing semi-quantitative reverse transcription coupled to PCR amplification we found that in addition to Prdm4 and Prdm5, Prdm2 and Prdm10 genes are expressed in hCECs. We further found that cultured primary hCECs or immortalised HCEC-12 cells express all of the Prdm genes found in CECs, but also express additional Prdm transcripts. This difference is most pronounced between Prdm gene expression patterns of CECs isolated from healthy human corneas and immortalised HCEC-12 cells. We further investigated Prdm 4 and Prdm 5 protein expression in cultured primary hCECs and HCEC-12 cells as well as in a human cadaveric whole cornea. Both Prdm 4 and Prdm 5 are expressed in human corneal endothelium, primary hCECs and in HCECs-12 cells, characterised by expression of the Na + /K + -ATPase. We observed that both proteins exhibit cytosolic (intracellular, but non-nuclear and distinct from extracellular fluid) as well as nuclear localisation within the endothelial layer, with Prdm 5 being more concentrated in the nuclei of the endothelial cells than Prdm 4. Thus, our work identifies novel Prdm genes specifically expressed in corneal endothelial cells which may be important in the control of CEC differentiation and proliferation., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: To characterize changes in the energy-producing metabolic activity and morphologic ultrastructure of corneal endothelial cells associated with diabetes mellitus., Methods: Transplant suitable corneoscleral tissue was obtained from donors aged 50 to 75 years. We assayed 3-mm punches of endothelium-Descemet membrane for mitochondrial respiration and glycolysis activity using extracellular flux analysis of oxygen and pH, respectively. Transmission electron microscopy was used to assess qualitative and quantitative ultrastructural changes in corneal endothelial cells and associated Descemet membrane. For purposes of analysis, samples were divided into four groups based on a medical history of diabetes regardless of type: (1) nondiabetic, (2) noninsulin-dependent diabetic, (3) insulin-dependent diabetic, and (4) insulin-dependent diabetic with specified complications due to diabetes (advanced diabetic)., Results: In total, 229 corneas from 159 donors were analyzed. Insulin-dependent diabetic samples with complications due to diabetes displayed the lowest spare respiratory values compared to all other groups (P ≤ 0.002). The remaining mitochondrial respiration and glycolysis metrics did not differ significantly among groups. Compared to nondiabetic controls, the endothelium from advanced diabetic samples had alterations in mitochondrial morphology, pronounced Golgi bodies associated with abundant vesicles, accumulation of lysosomal bodies/autophagosomes, and focal production of abnormal long-spacing collagen., Conclusions: Extracellular flux analysis suggests that corneal endothelial cells of donors with advanced diabetes have impaired mitochondrial function. Metabolic findings are supported by observed differences in mitochondrial morphology of advanced diabetic samples but not controls. Additional studies are needed to determine the precise mechanism(s) by which mitochondria become impaired in diabetic corneal endothelial cells.

Purpose: To explore the role of Rho-associated kinases (ROCK) in corneal physiology and regeneration, and the effects of suppressing its activity in stimulating corneal endothelial cell proliferation and migration in vitro and in vivo., Methods: Immunohistochemistry was performed to detect RhoA and ROCK-1 and ROCK-2 in human corneal tissue. Adult porcine corneal endothelial cells (CECs) were isolated, grown to confluence, and further characterized. Under the treatment of ROCK inhibitors, changes in the cellular distribution profile of ZO-1 and F-actin were examined by immunofluorescence staining. Corneal endothelial cells migration was evaluated by scratch assay and analyzed with Axiovision software. Cell proliferation was quantified using Click-iT EdU HCS Assay. In vivo, the corneal endothelia of rabbits were surgically injured and H-1152 was topically applied for 10 days. Progress of wound healing was evaluated daily by monitoring corneal edema, inflammation, and thickness using slit-lamp examination, photography, and pachymetry. Rabbits were euthanized and enucleated for further evaluation., Results: H-1152 exhibited significant stimulatory effect on CEC migration and proliferation in vitro compared with both untreated and Y-27632-treated cells. Furthermore, topical administration of H-1152 led to marked reduction in corneal edema and formation of multinucleate CECs in vivo suggestive of proliferation associated with healing., Conclusions: H-1152 exhibited a better stimulatory effect on CEC migration and proliferation in vitro than Y-27632. Our findings suggest that topical administration of H-1152 promotes healing of injured corneal endothelium in vivo. These results demonstrate the efficacy of ROCK inhibitors as a potential topical therapy for patients with corneal endothelial disease.

Purpose: Human chromosomes are protected at their end by a long portion of hexameric tandem repeats, the telomere. In somatic cells, telomere attrition caused by endogenous and exogenous oxidative stress as well as DNA replication can threaten genomic integrity and lead to the deterioration of tissue functions and an age-related physiological decline. The human eye is a complex organ in which cells of different ocular tissues are exposed to photo-oxidation, high mitochondrial metabolic activity, and/or replicative pressure., Methods: We employed a highly sensitive quantitative PCR technique to determine relative telomere length in different human ocular structures., Results: The longest telomeres in all ocular structures analyzed are found in neural retina, and the shortest are in the cornea. Within the retina, retinal pigment epithelium has four times shorter telomeres when compared to neural retina. However, no age-dependent telomere attrition in the retina and no difference between telomere lengths in the macular region and the rest of the retina have been found. In the cornea, stroma has the longer telomeres. In the corneal endothelium, we found a clear age-dependent telomere shortening. Since the endothelium is one of the most metabolically active ocular structure, this result suggests that endogenous oxidative stress from high mitochondrial activity is a major determinant of telomere loss in this structure., Conclusions: Taken together, our results imply that the aging process and telomere attrition in the different ocular structures are the result of multiple factors and could not be attributed to solely exogenous or endogenous oxidation or DNA replication.

Purpose: To clarify whether cultured human corneal endothelial cells (cHCECs), heterogeneous in their differentiation state, exhibit distinctive energy metabolism with the aim to develop a reliable method to sort cHCECs applicable for regenerative medicine., Methods: The presence of cHCEC subpopulations (SPs) was verified via surface cluster-of-differentiation (CD) marker expression. Cultured HCEC metabolic extracts or corresponding culture supernatants with distinctive cellular phenotypes in regard to energy-metabolism-related functional markers c-Myc and CD44 were prepared and analyzed via capillary electrophoresis-tandem mass spectrometry. The metabolic requirements of heterogeneous SPs of cHCECs were also investigated., Results: After successfully discriminating SPs, as verified via surface CD markers in terms of their secretory metabolites, we found that the CD44+++ SP with cell-state transition (CST) exhibited disposition for anaerobic glycolysis, whereas the CD44-SP without CST was disposed to mitochondria-dependent oxidative phosphorylation (OXPHOS). These results raised the possibility of establishing effective culture conditions to selectively expand mature cHCECs with a hexagonal cobblestone shape and inclination for mitochondria-dependent OXPHOS., Conclusions: The findings of this study open a pathway for monitoring the disposition of cHCECs via their energy metabolism, thus leading to safe and stable regenerative medicine by use of metabolically defined cHCECs in cell-suspension form.

Purpose: Human corneal endothelial cell (HCEC) density decreases with age, surgical complications, or disease, leading to vision impairment. Such endothelial dysfunction is an indication for corneal transplantation, although there is a worldwide shortage of transplant-grade tissue. To overcome the current poor donor availability, here we isolate, expand, and characterize HCECs in vitro as a step toward cell therapy., Methods: Human corneal endothelial cells were isolated from cadaveric corneas and expanded in vitro. Cell identity was evaluated based on morphology and immunocytochemistry, and gene expression analysis and flow cytometry were used to identify novel HCEC-specific markers. The functional ability of HCEC to form barriers was assessed by transendothelial electrical resistance (TEER) assays., Results: Cultured HCECs demonstrated canonical morphology for up to four passages and later underwent endothelial-to-mesenchymal transition (EnMT). Quality of donor tissue influenced cell measures in culture including proliferation rate. Cultured HCECs expressed identity markers, and microarray analysis revealed novel endothelial-specific markers that were validated by flow cytometry. Finally, canonical HCECs expressed higher levels of CD56, which correlated with higher TEER than fibroblastic HCECs., Conclusions: In vitro expansion of HCECs from cadaveric donor corneas yields functional cells identifiable by morphology and a panel of novel markers. Markers described correlated with function in culture, suggesting a basis for cell therapy for corneal endothelial dysfunction.

Corneal transparency is dependent on the maintenance of the structural integrity and functional activity of its epithelial and endothelial limiting layers and the stroma. Different transient receptor potential (TRP) channel subtypes are expressed in cells and on corneal sensory nerve endings. They serve as sensors and transducers of environmental stimuli that can reduce tissue transparency. These nonselective cation channels are members of a superfamily sharing TRP box protein sequence homology having 6 membrane spanning domains with a pore between the fifth and sixth segments. TRP channels are composed of 4 monomeric subunits that oligomerize in homomeric or heteromeric configurations derived from different TRP subtypes belonging to the same or any of 6 different subfamilies. TRP subfamily members identified in the cornea include those belonging to the canonical, vanilloid, ankyrin, or melastatin subfamilies. In this review, we specifically focus on the functional roles of TRPV1 and TRPA1 expression in the cornea as their activation provides adaptive nociceptive and immune responses to noxious environmental stresses such as irritating ligands, temperature fluctuations, rises in ambient osmolarity, mechanical stretch, decline in pH, and tissue injury. Our previous studies have indicated that TRPV1 and TRPA1 subtypes are potential drug targets for improving corneal wound healing after alkali burns, because injury-induced fibrosis, neovascularization, and inflammation in either TRPV1 or TRPA1 gene-silenced mice were all significantly reduced.

Purpose: Cultured human corneal endothelial cells (HCECs) are anticipated to serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction. However, corneal endothelial cells (CECs) tend to exhibit fibroblastic transformation, thereby losing their functional phenotype when cultured. The purpose of this study was to investigate the usefulness of surface markers of CECs displaying fibroblastic phenotypes as a means of cell characterization., Methods: The expression levels of 242 cell surface antigens were screened in cultured human and monkey CECs using flow cytometry. An expression intensity ratio of nonfibroblastic/fibroblastic CECs > 2 and of fibroblastic/nonfibroblastic CECs > 2 were selected as indicating nonfibroblastic and fibroblastic markers, respectively. Nonfibroblastic and fibroblastic CECs were mixed, and CD73-positive and -negative cells were sorted using flow cytometry and further cultured. The functional phenotype of the sorted cells was evaluated according to morphology and the expression of function-related (Na(+)/K(+)-ATPase and ZO-1) and fibroblastic (type I collagen and fibronectin) markers., Results: Flow cytometry analysis demonstrated that CD98, CD166, and CD340 are elevated in HCECs of nonfibroblastic phenotype, while CD9, CD49e, CD44, and CD73 are markers of fibroblastic phenotype HCECs. The CECs that sorted as CD73-negative exhibited normal hexagonal morphology and expressed functional markers, whereas CECs that sorted as CD73-positive exhibited the fibroblastic phenotype., Conclusions: These markers will be useful for quality control to characterize the phenotype of cells destined for tissue engineering-based therapy. In addition, this selection protocol will provide a novel method for purification of functional cells., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

The corneal endothelium (CE) is a single layer of cells lining the posterior face of the cornea providing metabolic functions essential for maintenance of corneal transparency. Adult CE cells lack regenerative potential, and the number of CE cells decreases throughout life. To determine whether endogenous DNA damage contributes to the age-related spontaneous loss of CE, we characterized CE in Ercc1(-/Δ) mice, which have impaired capacity to repair DNA damage and age prematurely. Eyes from 4.5- to 6-month-old Ercc1(-/Δ) mice, age-matched wild-type (WT) littermates, and old WT mice (24- to 34-month-old) were compared by spectral domain optical coherence tomography and corneal confocal microscopy. Histopathological changes in CE were further identified in paraffin tissue sections, whole-mount immunostaining, and scanning electron and transmission electron microscopy. The CE of old WT mice displayed polymorphism and polymegathism, polyploidy, decreased cell density, increased cell size, increases in Descemet's thickness, and the presence of posterior projections originating from the CE toward the anterior chamber, similar to changes documented for aging human corneas. Similar changes were observed in young adult Ercc1(-/Δ) mice CE, demonstrating spontaneous premature aging of the CE of these DNA repair-deficient mice. CD45(+) immune cells were associated with the posterior surface of CE from Ercc1(-/Δ) mice and the tissue expressed increased IL-1α, Cxcl2, and TNFα, pro-inflammatory proteins associated with senescence-associated secretory phenotype. These data provide strong experimental evidence that DNA damage can promote aging of the CE and that Ercc1(-/Δ) mice offer a rapid and accurate model to study CE pathogenesis and therapy., (© 2013 the Anatomical Society and John Wiley & Sons Ltd.)

Purpose: Sulfur mustard (SM) is a highly reactive vesicant that causes severe ocular injuries. Following exposure to moderate or high doses, a subset of victims develops a chronic injury known as mustard gas keratopathy (MGK) involving a keratitis of unknown etiopathogenesis with secondary keratopathies such as persistent epithelial lesions, corneal neovascularization, and progressive corneal degeneration. This study was designed to determine whether SM exposure evokes acute endothelial toxicity and to determine whether endothelial pathologies were specifically observed in MGK corneas as opposed to healed corneas., Methods: Corneas of New Zealand white rabbits were exposed to SM vapor, and the corneal endothelium was evaluated at 1 day and 8 weeks using scanning electron microscopy (SEM), transmission electron microscopy (TEM), in vivo confocal microscopy (IVM), and fluorescent microscopy. Barrier function was measured by uptake of a fluorescent dye injected into the anterior chamber., Results: A centripetal endothelial injury at 1 day was observed by SEM, TEM, IVM, and fluorescent microscopy. In vivo confocal microscopy revealed additional cytotoxicity between 1 and 13 days. In contrast to healed corneas, which appeared similar to sham-exposed naive eyes at 8 weeks, MGK corneas exhibited significant evidence of continued pathological changes in the endothelium., Conclusions: Endothelial toxicity occurs at the right time and with the appropriate pathophysiology to contribute to MGK. Based on these findings, we propose a model that explains the relationships among SM dose, the biphasic progression, and the various clinical trajectories of corneal SM injury and that provides a mechanism for temporal variations in MGK onset. Finally, we discuss the implications for the management of SM casualties.

Purpose: To investigate the protective effect of rapamycin on oxidative stress-induced cell death of human corneal endothelial cells (HCECs)., Methods: HCECs were cultured according to previously published methods. With treatment of 0 mM or 5 mM of tert-butyl hydroperoxide (tBHP) with various concentrations (0, 25 and 50 nM) of rapamycin, reactive oxygen species (ROS) production was measured using an oxidation-sensitive fluorescent probe, 2'7'-dichlorofluorescin diacetate (DCFH-DA, USA) methods. Cell viability was assayed by the method of Cell Counting Kit-8 (CCK-8, Wako). The levels of cellular glutathione were also assessed enzymatically with glutathione reductase by using a commercial glutathione (GSH) assay kit (Cayman Chemical, USA)., Results: Rapamycin reduced 2'7'-dihydrodichlorofluorescein oxidation and increased GSH in HCECs. Rapamycin significantly inhibited tBHP-induced ROS production. Cells treated with rapamycin showed higher viability compared to control at 5 mM tBHP. Rapamycin effectively protected HCECs from ROS-induced cell death through increasing intracellular GSH., Conclusion: Our data suggest that rapamycin protects HCECs from oxidative injury-mediated cell death via inhibition of ROS production and enhancement of GSH.

One of the most important steps during Descemet stripping automated endothelial keratoplasty (DSAEK) is attaching the donor Descemet lenticule to the host's cornea. This is usually carried out by injecting air into the anterior chamber with a syringe and needle. We present an alternative technique using an air fluid exchange system connected to an anterior chamber maintainer used in 22 cases in our institute. This technique has proven to be simple and fast, thus facilitating the successful performance of DSAEK surgery., (Copyright © 2011 Canadian Ophthalmological Society. Published by Elsevier Inc. All rights reserved.)

Background: The purpose of the experiment reported here was to assess the involvement of advanced glycation end products (AGEs), oxidative stress, and nuclear factor kappa-B (NF-κB) activation in the development of diabetic keratopathy., Methods: Diabetes was induced by intraperitoneal streptozotocin injection in male Sprague-Dawley rats. The thickness of the cornea was measured. Apoptosis was detected by TUNEL assay and western blot for caspase-3. The expression of AGEs and 8-hydroxydeoxyguanosine (8-OHdG) were studied by immunohistochemistry in corneal tissues of normoglycaemic and diabetic rats. NF-κB activation was evaluated by electrophoretic mobility shift assay and southwestern histochemistry., Results: Corneal edema was observed in diabetic rats. The thickness of cornea was higher in diabetic than in control rats. AGEs were accumulated in corneal tissues. 8-OHdG and NF-κB were identified in corneal epithelium, stroma and endothelium, and its expressions were greater in diabetic than in those of control rats. Diabetes induces significant alterations in rat corneal tissue structure., Conclusions: The higher expression of AGE, 8-OHdG and NF-κB in corneal tissues of diabetic rats suggests that these factors are involved in apoptosis and in subsequent corneal alterations related to diabetic keratopathy.

Purpose: To determine through the use of immunohistochemistry the origins of retrocorneal cellular and fibrillar membranes., Design: Retrospective, clinicopathologic study using surgically removed human corneal tissues., Methods: Clinical records of patients' ocular diseases and surgical procedures were reviewed. Immunohistochemical staining was performed on 5 enucleated control globes, 32 penetrating keratoplasty specimens, and 6 Descemet stripping endothelial keratoplasty specimens to analyze: (1) the normal corneal epithelium, stroma, and endothelium; and (2) stromal scars, endothelial abnormalities, and retrocorneal membranes. Paraffin sections were stained with hematoxylin and eosin, periodic acid-Schiff, and Masson trichrome methods, and immunohistochemical analyses were performed with commonly available monoclonal and polyclonal antibodies for various cytokeratins (CKs), CD34, alpha-smooth muscle actin (SMA), and vimentin., Results: Five subtypes among 28 retrocorneal membranes were characterized. Twelve fibrous (keratocytic) membranes of stromal origin had coarse collagen and immunostained negatively for all CKs, but strongly for vimentin and alpha-SMA, the last the only marker of diagnostic value. Nine metaplastic endothelium-derived membranes produced delicate collagenous matrices and immunoreacted with CK7, vimentin, and alpha-SMA. Two epithelial multilaminar or monolaminar membranes reacted with CK cocktail and wide-spectrum CK, mildly with CK7 (not observed in orthotopic surface epithelium), and negatively for alpha-SMA and vimentin. The final 2 categories were indeterminate or non-immunoreactive (3 specimens) and mixed (2 specimens)., Conclusions: Immunohistochemistry can diagnose retrocorneal membranes of different provenances reliably in most cases. Clinical correlations established that these membranes develop after serious inflammatory disorders, prolonged wounding or ulcerations, and multiple surgeries (an average of 3.4 per patient)., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Gene therapy of the cornea shows promise for modulating corneal transplant rejection but the most appropriate vector for gene transfer has yet to be determined. We investigated a lentiviral vector (LV) for its ability to transduce corneal endothelium. A lentivector expressing enhanced yellow fluorescent protein (eYFP) under the control of the Simian virus type 40 early promoter (LV-SV40-eYFP) transduced 80-90% of rat, ovine and human corneal endothelial cells as detected by fluorescence microscopy. The kinetics of gene expression varied among species, with ovine corneal endothelium showing a relative delay in detectable reporter gene expression compared with the rat or human corneal endothelium. Vectors containing the myeloproliferative sarcoma virus promoter or the phosphoglycerate kinase promoter were not significantly more effective than LV-SV40-eYFP. The stability of eYFP expression in rat and ovine corneas following ex vivo transduction of the donor cornea was assessed following orthotopic corneal transplantation. Following transduction ex vivo, eYFP expression was maintained in corneal endothelial cells for at least 28 days after corneal transplantation in the sheep and >60 days in the rat. Thus, rat, ovine and human corneal endothelial cells were efficiently transduced by the LV, and gene expression appeared stable over weeks in vivo.

Background: Ocular disorders associated with the human immunodeficiency virus are numerous as are ocular side effects from medications used to treat all of the manifestations of the virus. This report presents a unique case of bilateral, peripheral, and corneal endothelial deposits that may be a result of either the human immunodeficiency virus or the medication rifabutin. Rifabutin was the only medication prescribed that is known to cause endothelial deposits. Rifabutin is part of a multidrug therapy to prevent or treat Mycobacterium avium complex, a common pulmonary disease of immunocompromised individuals., Case Report: A 69-year-old man with a 20-year history of being human immunodeficiency virus-positive presented with bilateral, asymptomatic, peripheral, and corneal endothelial deposits of unknown etiology. Literature research suggested that the deposits did not appear like cytomegalovirus retinitis-related deposits but rather a variant of rifabutin-associated deposits., Conclusions: These rifabutin-associated deposits differed from known rifabutin-associated deposits previously reported in the literature. These deposits have increased in pigmentation and density 5 years after the patient discontinued the drug. This case may represent another variation of rifabutin-associated endothelial deposits. Knowledge of human immunodeficiency virus and all the associated ocular findings (owing to both the condition and its treatment) is important, because the length of time patients are living with human immunodeficiency virus is increasing.

Purpose: To establish a method for the mass production of human corneal endothelium (HCE) precursors and the therapeutic application of these cells in a rabbit CE-deficiency model., Methods: A sphere-forming assay was performed to produce precursors from cultured HCE. Various marker expressions were examined in the sphere colonies, and their progenies by immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The transport activity of the sphere-derived cell sheet was evaluated by the Ussing chamber system. DiI-labeled precursors obtained from cultured HCE were injected the anterior chamber of the eye in a rabbit CE-deficiency model, and the eye-down position was maintained for 24 hours for attachment to Descemet's membrane (sphere eye-down group). The sphere eye-down and control groups, observed for 28 days after surgery, underwent histologic and fluorescence microscopic examinations., Results: Cultured HCE formed primary and secondary sphere colonies. The spheres expressed alpha-smooth muscle actin and nestin, and progeny expressed alpha-smooth muscle actin, confirmed by RT-PCR. The progeny showed an HCE-like hexagonal shape, were confluent, and had adequate transport activity. Mean corneal thickness in the sphere eye-down group was significantly less than in the other control groups 14, 21, and 28 days (P < 0.006) after surgery. The HCE-like hexagonal cells detected on the Descemet's membrane are DiI-positive in the sphere eye-down group., Conclusions: The findings demonstrate that culture of HCE can promote mass production of HCE precursors, determined by sphere-forming assay. Injection of precursors derived from cultured HCE into the anterior chamber is an effective treatment strategy for CE deficiency in a rabbit model.

Purpose: To isolate precursor cells derived from rabbit corneal endothelium (CE) and to use them for the treatment of CE deficiency in a rabbit model., Methods: A sphere-forming assay was performed to isolate precursor cells from rabbit CE. Immunocytochemistry was used to examine marker expressions of neural and mesenchymal cells in the sphere colonies and their progenies. The pump function of the CE sheet was evaluated by measurement of the potential difference and short circuit current. Precursors obtained from rabbit CE by a sphere-forming assay were injected into the anterior chamber of the eye, after which an eye-down (i.e., CE up) position was maintained for 24 hours to allow attachment by gravitation (sphere eye-down group). The sphere eye-down and control groups, observed for 28 days after surgery, underwent histologic and fluorescence microscopic examinations., Results: Rabbit CE formed primary and secondary sphere colonies. The progeny expressed alpha-smooth muscle actin, nestin, and neural markers and showed a CE-like hexagonal shape and adequate transport activity. Mean corneal thickness in the sphere eye-down group was significantly less than in the other control groups 3, 7, 14, 21, and 28 days (P < 0.05) after surgery. CE-like hexagonal cells were detected on Descemet's membrane, and corneal edema was substantially suppressed. DiI-labeled cells were spread over the rear corneal surface in the sphere eye-down group only., Conclusions: Precursors from rabbit CE were isolated by a sphere-forming assay. Rabbit CE-derived sphere therapy is an effective treatment in a rabbit CE deficiency model.

Free-radical formation may play a role in postoperative complications of phacoemulsification (e.g., corneal endothelium damage from mechanical injury). The present experiments were aimed at investigating whether different molecular weight ranges (2000-2600, 2600-3200, or 3200-3800 kDa) of hyaluronan may influence free radical formation, corneal endothelium damage, and inflammation parameters after phacoemulsification in the rabbit eye. The viscoelastic substance was injected in the anterior chamber of rabbits' eyes before phacoemulsification, at a 2.5% concentration. The formation of free radicals was determined by adding luminol to the irrigation media and measuring the chemoluminescence in eyes. The corneal endothelial damage was evaluated by measuring the corneal central thickness by pachimetry. The inflammation parameters were measured by calculation in aqueous humor of peak levels of leukocytes and prostaglandin E(2) (PGE(2)) and evaluation in uveal tissue of myeloperoxidase activity. Hyaluronan decreased by about 58-60% free-radical formation during phacoemulsification, reduced by about 76-80% modifications in mean corneal thickness and by about 54-61% the corneal endothelial cell loss in all molecular weight ranges used. No difference was found among various molecular weight ranges. The highest molecular weight range showed to be more potent than the lowest range for reduced number of inflammation cells and level of PGE(2) in aqueous humor. Thus, hyaluronan reduces free-radical formation, exerts protection on the corneal endothelium and exerts anti-inflammation properties after phacoemulsification in rabbits. The latter effect seems to depend on the molecular weight of the substance.

Purpose: To compare corneal endothelial bleb formation and disappearance processes between rigid gas-permeable (RGP) contact lenses and soft contact lenses (SCL) in the same class of oxygen transmissibility (Dk/L)., Methods: Experiments were conducted on 20 eyes of 10 nonwearers of contact lenses using six kinds of RGP lenses or soft lenses with hyper (A), high (B), and medium (C) classes of Dk/L. Each subject kept their eyes closed for 20 minutes after putting on each test lens. Starting just after eye opening, the subjects' eyes were examined for blebs every 5 minutes by specular microscopy, and percentage area of the blebs were calculated. For control, the same eyes were also examined without contact lens wear., Results: The percentage area of the blebs just after eye opening was 1.2% for RGP-A, 1.7% for SCL-A, 5.1% for RGP-B, 4.7% for SCL-B, 6.9% for RGP-C, 8.6% for SCL-C, and 0.4% for the control. The difference from the control became insignificant in the bleb disappearance process just after eye opening for RGP-A and SCL-A, after 5 minutes for RGP-B and SCL-B, after 10 minutes for RGP-C, and after 30 minutes for SCL-C., Conclusions: Regarding bleb formation and disappearance, there was no difference in influence on the corneal endothelium between RGP lenses and soft lenses with similar Dk/L in the high and hyper classes. In the medium class, however, it was suggested that RGP lenses may have less of an impact on the corneal endothelium than soft lenses do.

The purpose of this study was to identify the in vivo microstructural characteristics of an animal model of 'epithelialization of the endothelium' that are similar in appearance to the in vivo confocal microscopical appearance of corneal endothelium previously thought to be diagnostic of irido-corneal endothelial syndrome, and correlate these observations with ex vivo in situ confocal microscopical analysis. A rat model (n = 8 eyes)of transient 'epithelialization of the endothelium' resulting from superficial corneal trauma, was developed and analysed using in vivo confocal microscopy. One animal was killed at 48 hand the cornea was immuno-labelled and analysed, using ex vivo in situ confocal digital image reconstruction. Reversible 'epithelialization of the endothelium' was observed by in vivo confocal microscopy 48 h after superficial corneal trauma in all eight eyes. Ex vivo in situ analysis failed to demonstrate immunohistological characteristics of epithelialization. In vivo confocal microscopy is based on optical principles, and as a result various structural alterations may present with apparently identical characteristics that should be interpreted cautiously, on the basis of the presented clinicopathological observations.

Objective: To evaluate tear production, corneal topography, accuracy of refractive correction, and best spectacle-corrected visual acuity in eyes that had moderate to severe rose bengal staining develop on the flap compared with eyes with little or no staining on the flap, the first few months after laser in situ keratomileusis (LASIK). None of the eyes in this study had significant preoperative dry eye disease., Design: Retrospective case control study., Participants: Individual eyes of 19 consecutive patients with moderate to severe punctate epithelial erosions and rose bengal staining on the flap 1 to 3 months after LASIK were compared with eyes of 19 concurrent patients who did not have punctate epithelial erosions or more than trace staining on the flap develop., Methods: Nonparametric statistical analyses were used to compare tear secretion, corneal topographic irregularity, spherical equivalent, and visual acuity 3 and 6 months after surgery. Some eyes in both groups also had analysis of tear secretion 1 month after surgery., Main Outcome Measures: Schirmer's test without anesthesia, the topographic corneal irregularity measurement (CIM), the difference between attempted and achieved spherical equivalent, and the loss of best spectacle-corrected visual acuity., Results: There was no difference in tear production 1, 3, or 6 months after LASIK in patients who had punctate epithelial erosions and rose bengal staining on the flap develop and those who did not. There was no significant difference in the CIM or mean accuracy of the refractive correction in the two groups, but some patients had a transient decrease in best spectacle-corrected visual acuity. Flap rose bengal staining resolved by 6 months after LASIK in most affected patients., Conclusions: LASIK-induced rose bengal staining in patients without preexisting dry eye is likely neurotrophic epitheliopathy, because there is no difference in mean tear production between patients who have significant punctate epithelial erosions and rose bengal staining develop on the flap and those who do not. The signs and symptoms of LASIK-induced (presumed) neurotrophic epitheliopathy tend to resolve approximately 6 months after surgery. This disorder tends to be more common and severe in patients with pre-existing dry eye disease.

Aims: To investigate the ultrastructural localisation of proteoglycans (PG), betaig-h3 (keratoepithelin), tenascin-C (TN-C)), fibrillin, and fibronectin in bullous keratopathy (BK) corneas., Methods: Five corneas from cases of pseudophakic bullous keratopathy (BK) were examined by electron microscopy. PG were demonstrated using cuprolinic blue, and the proteins betaig-h3, TN-C, fibrillin, and fibronectin were immunolocalised with rabbit anti-betaig-h3, mouse anti-TN-C (BC10 and TN2), mouse anti-fibrillin-1 (MAB2502), mouse anti-fibrillin (MAB1919), and rabbit anti-fibronectin by using a standard immunogold technique., Results: Epithelial cells contained numerous vacuoles. Epithelial folds and large, electron lucent subepithelial bullae were present. Basal lamina was thickened and traversed by disrupted anchoring filaments. In the stroma, interfibrillar collagen spacing was increased and abnormally large PG were present. Descemet's membrane (DM) contained lucent spaces in which there were small filaments. Keratocyte and endothelial cells contained melanin granules. A posterior collagenous layer (PCL) contained numerous microfilaments and wide spacing collagen fibres with a periodicity of 100 nm. Large quantities of abnormal PG were observed at the endothelial face of the PCL. Very strong labelling with betaig-h3 antibody was observed in the basement membrane, Bowman's layer, stroma, DM, and PCL, but not in keratocytes and endothelial cells. Strong labelling with BC10 and TN2 was seen below the epithelium, in electron lucent spaces where the hemidesmosomes were absent, in the fibrotic pannus, in parts of Bowman's layer, the stroma, and Descemet's membrane. Labelling with BC10 was stronger and more evenly distributed than with TN2. Fibrillin-1 (MAB2502) and fibrillin (MAB1919) labelling was similar to TN-C labelling. Fibrillin (MAB1919) labelling was stronger than fibrillin-1 (MAB2502) labelling., Conclusions: Immunoelectron microscopy showed precise labelling of proteins at both the cellular and the subcellular level. Expression of proteins betaig-h3, TN-C, fibrillin, and fibronectin was highly increased compared with normal cornea. In the oedematous stroma, increased collagen fibril separation may facilitate a wider distribution of some soluble proteins, such as betaig-h3, throughout stroma. The modified expression of the proteins studied in these cases of BK may be regarded as part of an injury response.

Purpose: To study the corneal endothelial morphological changes and endothelial barrier function in contact lens wearers., Methods: Specular microscopy and anterior segment fluorophotometry were performed on 116 controls (group 1) and 76 daily wear soft contact lens wearers. Group 2 patients (n = 34) had been wearing contact lenses for less than 5 years and group 3 (n = 42) for more than 5 years. The relationship of corneal thickness, endothelial cell density, hexagonal cell percentage, coefficient of variation in cell area, corneal autofluorescence and corneal endothelial permeability to the contact lens wear duration was studied., Results: The average corneal thickness of contact lens wearers did not differ significantly from controls (0.533 +/- 0.031, 0.538 +/- 0.044, 0.532 +/- 0.031 mm for groups 1, 2 and 3, respectively; p = 0.89). However, there was a significant tendency toward corneal thinning with a longer history of contact lens wear (r = -0.31, p = 0.002 in groups 2 and 3). The percentage of hexagonal cells decreased with both increasing hours of contact lens wear per day (r = -0.36, p < 0.001) and the duration of contact lens wear in years (r = -0.33, p < 0.001), but there was no significant change in endothelial cell density in contact lens wearers. The coefficient of variation in cell size correlated more closely with increased hours of contact lens wear per day (r = 0.35, p = 0.002) than with the duration of contact lens wear in years (r = 0.12; p = 0.31). In the functional study, corneal autofluorescence increased in contact lens wearers (5.13 +/- 0.71 ng/ml in group 1, 6.45 +/- 2.03 ng/ml in group 2 and 7.21 +/- 1.51 ng/ml in group 3, respectively, p < 0.001) and the mean endothelial permeability decreased in contact lens wearers (3.89 +/- 0.95 x10(-4)/cm in group 1, 2.71 +/- 0.73 x 10(-4)/cm in group 2 and 2.95 +/- 0.91 x 10(-4)/cm in group 3, respectively, p = 0.003)., Conclusions: Daily wear soft contact lenses caused morphological changes in the corneal endothelium. With an increasing span of contact lens wear, there was a significantly increased variation in cell size, a decreased hexagonal cell percentage, an evident intercellular dark area and rosette formation. The corneal autofluorescence increased and the overall endothelial permeability decreased as a consequence of contact lens wear. Contact lens wear also caused corneal thinning, and the cornea became thinner with increasing duration of contact lens wear., (Copyright 2001 S. Karger AG, Basel)

Purpose: This study was undertaken to evaluate the endothelial pump function by monitoring both corneal swelling response under hypoxia and dehydration response following hypoxia in vivo., Methods: Humidified nitrogen gas was used to obtain corneal swelling, and humidified gas mixed with oxygen and nitrogen was used for corneal dehydration. First, in 6 young volunteers, we investigated the most suitable oxygen level for evaluating pump function by changing oxygen levels. Then, with the optimal oxygen level, we attempted to evaluate pump function in 53 normal subjects, 5 Fuchs' dystrophy patients, and 3 iridocorneal endothelial syndrome (ICE) patients., Results: Swelling rate showed similar values regardless of age, but both dehydration rate and swelling rate plus dehydration rate decreased with aging. The swelling rate of 5 guttata corneas was significantly higher than that of age-matched control corneas. In contrast, dehydration rate markedly decreased in guttata corneas, while the swelling rate plus dehydration rate of guttata corneas was comparable to that of age-matched corneas. In the 3 ICE corneas, however, swelling rate, dehydration rate, and swelling rate plus dehydration rate were markedly lower than those of both the fellow corneas and the age-matched control corneas., Conclusion: These observations lead us to conclude that in order to evaluate pump function, it is necessary to monitor not only dehydration response following hypoxia but also swelling response under hypoxia.

Purpose: To present unusual cases with corneal graft deposits and discuss eye bank criteria concerning donors treated with phenothiazines., Methods: We examined two patients with unusual corneal graft deposits after uneventful corneal transplantation. Donors' files and eye bank procedures were reviewed., Results: Fine corneal endothelial punctate deposits were detected in both corneal grafts and did not resolve in the following 18 months., Conclusion: To our knowledge, this is the first case report documenting transplantation of corneas with chlorpromazine deposits. Delicate corneal findings can be missed before transplantation.

Purpose: To test the hypothesis that the aberrant, cytokeratin-expressing cells that replace endothelium in the iridocorneal endothelial (ICE) syndrome are of endothelial origin., Methods: Corneas from four patients with Chandler's syndrome and three with essential iris atrophy were examined by two-color immunofluorescence for simultaneous expression of cytokeratins and two markers of endothelial lineage: vimentin and the antigen recognized by the antiendothelial monoclonal antibody 2B4.14.1., Results: In six corneas, unequivocal endothelial staining for cytokeratins was present; in each of these, cells coexpressing cytokeratins and the two endothelial markers were clearly identifiable. In the remaining cornea, weak cytokeratin staining that colocalized with vimentin was present., Conclusions: These results lend strong support to the hypothesis that the "epithelial-like" endothelial cells in ICE syndrome are cells of endothelial lineage rather than heterotopia of epithelial cells; these cells probably arise via a metaplastic transformation of preexisting endothelium.

Aims: This study was undertaken to confirm the distribution and expression of the molecule CD44 on human corneas under normal and pathological conditions., Methods: Fifty eight corneal buttons from adult patients suffering from various corneal diseases and four normal corneas were included in this study. Frozen sections were stained immunohistochemically with monoclonal antibodies against human CD44 using an APAAP method and observed under a light microscope., Results: In normal corneas CD44 was predominantly expressed on the membranes of basal epithelial cells and on the keratocytes, as well as on the vascular endothelial cells of the corneal limbi, but was not expressed on corneal endothelial cells. Enhanced expression of CD44 was observed on the epithelium of corneas with inflammation and allograft rejection. In a number of abnormal conditions including allograft rejection, corneal trauma, primary and secondary corneal endothelial decompensation the remaining endothelial cells stained positively for CD44. However, in some corneas of keratitis, keratoconus, and dystrophy the endothelium which appeared relatively integral in morphology and amount remained CD44 negative., Conclusions: These results suggest that CD44, the hyaluronate receptor, may play an important role in corneal cell-cell and cell-matrix interactions. Its regulation is closely related to corneal inflammatory reactions. The induction of CD44 on corneal endothelium might play a potential role in compensatory processes when corneal endothelial cells are injured.

Purpose: The iridocorneal-endothelial (ICE) syndrome is characterized by glaucoma, corneal failure, and iris destruction. Specular photomicroscopy of the corneal endothelium in this disease shows a population of abnormal cells named ICE-cells. Comparison between ultrastructural examination and specular photomicroscopy demonstrates that the histologic equivalent of ICE-cells are cells with an epithelial phenotype. The authors have studied the differentiation markers expressed by ICE-cells using an ultrastructural immunocytochemical technique., Methods: Seven keratoplasty specimens from patients with the ICE syndrome were examined by scanning and transmission electron microscopy and light and electron microscopic immunocytochemistry. Comparison was made with three normal corneas. Immunocytochemical studies were performed with monoclonal antibodies to broad-spectrum cytokeratins, cytokeratins 3, 5/8, 8/18 and 19, vimentin, and epithelial membrane antigen., Results: ICE-cells were morphologically similar to epithelial cells and expressed the same profile of differentiation markers as did normal limbal epithelial cells., Conclusions: ICE-cells may arise from an embryologic ectopia of ocular surface epithelium. Alternatively, these findings are consistent with a metaplastic stimulus resulting in a profound change in the phenotype of normal corneal endothelial cells.

Purpose: To evaluate immunohistochemical staining of the endothelia of corneas from patients with clinical diagnoses of iridocorneal endothelial (ICE) syndrome., Methods: Corneas diagnosed with ICE syndrome and removed during corneal transplantation were freshly frozen, sectioned, and stained with monoclonal antibodies to keratin subgroups, vimentin, desmin, and a series of other antibodies against intermediate filaments. Transmission electron microscopy was performed on segments of these corneas fixed in glutaraldehyde., Results: There was almost universal staining of the endothelial layer with A1 and A3 keratin monoclonal antibodies and vimentin. Transmission electron microscopy of the corneas also confirmed features consistent with keratin., Conclusions: The "endothelial" cell layer in the iridocorneal endothelial syndrome has electron microscopic and immunohistochemical characteristics of epithelial-like cells, but it also cross-reacts with vimentin, suggesting that these cells retain or derive some endothelial staining characteristics as well. This "epithelialization" of the endothelial layer may explain the progressive cellular proliferation across angle and iris similar to that seen in iatrogenic epithelial downgrowth and posterior polymorphous endothelial dystrophy.

The immunocytologic characteristics of two formalin-fixed, paraffin-embedded corneas from patients with the iridocorneal endothelial (ICE) syndrome and unaffected control corneas were studied. Binding of polyclonal antisera to Factor VIII, S-100 protein, involucrin, neuron specific enolase (NSE), and the lectins peanut agglutinin and Ulex europaeus agglutinin-1 was performed using the standard peroxidase-anti-peroxidase method. We detected reactive patterns of monoclonal antibodies to cytokeratins (34BE12 is a 56-58 kD mouse IgG reactive to stratified epithelia; Pkk1 is a 44-54 kD mouse IgG reactive to simple epithelia; and KL1 is a 55-57 kD mouse IgG reactive to epidermis and simple epithelia) using the standard avidin-biotin complex method. Staining properties were similar for the polyclonal antisera, lectins, NSE, and chromogranin in corneas with ICE syndrome and in the controls. However, the cytokeratins 34BE12, Pkk1, and KL1 were detected in the endothelium of the corneas with the ICE syndrome but not in the controls. These findings suggest that various cytokeratins are expressed in the corneal endothelium in the ICE syndrome that are not expressed in unaffected corneal endothelium.

Cell-matrix interactions play a fundamental role in normal and pathological conditions. They can be mediated by the cytoadhesin subgroup of the integrin superfamily of adhesion molecules. Its members include the vitronectin receptor (VNR) and the platelet glycoprotein IIb/IIIa (GP IIb/IIIa). Both receptors are composed of an alpha-chain (alpha v and alpha IIb, respectively) coupled to a beta 3-chain. Using in situ immunohistochemistry and monoclonal antibodies, the authors studied the distribution of GP IIIa (common beta 3-chain), GP IIb/IIIa (alpha IIb-chain) and VNR (alpha v-chain) in normal and pathological corneal tissues. In the normal cornea, the limbal vascular endothelium was weakly alpha v-positive. Occasionally, faint and granular staining was seen in the epithelium. In the pathological samples, an upregulated expression of the alpha v-chain was noticed on the corneal epithelium as well as on fibroblasts and corneal endothelium. The alpha IIb and beta 3-chains were consistently absent. These data suggest that expression of the VNR-alpha v-chain in the human cornea is modulated by soluble factors released during inflammation and wound healing. Dissociation of expression of the alpha v and beta 3-chains suggests usage of an alternative beta-chain by the VNR-alpha v-chain.

The clinical and specular microscopic results of 40 cases (39 patients) of penetrating keratoplasty during which a Kelman-style anterior chamber intraocular lens was implanted were reviewed retrospectively. Thirty-one pseudophakic eyes received an intraocular lens exchange and nine aphakic eyes received a secondary intraocular lens. Postoperative follow-up averaged 24.5 months (range, 3 to 51 months). At 1, 2, and 3 years after keratoplasty, 39.3%, 63.2%, and 63.6% of eyes, respectively, had visual acuities of 20/40 or better. Ninety-five percent of the grafts remained clear. Causes of poor postoperative visual acuity included cystoid macular edema (32.5%), new glaucoma (22.5%), and age-related macular degeneration (10.0%). Other causes were endothelial rejection leading to graft failure, corneal ulceration, and retinal detachment. Corneal endothelial cell loss by specular microscopy was 11.5% at 1 years, 21.3% at 2 years, and 25.0% at 3 years. These results were compared with cell loss associated with iris-sutured posterior chamber lenses in penetrating keratoplasty. Visual outcomes and complication rates were similar between these two methods; however, the endothelial attrition at 1 and 2 years for the sutured posterior chamber lens was greater than that of the Kelman anterior chamber lens.

The relationship of contact lens-induced edema to epithelial and endothelial function was determined in isolated superfused rabbit corneas. Placement of a polymethyl methacrylate (PMMA) contact lens on the cornea caused swelling rates of 15-28 microns/hr compared to 0-6 microns/hr in paired control corneas. The edema increased with temperature (P less than 0.01). PMMA-induced swelling was significant in: 1) bicarbonate-free Ringer's solution; 2) chloride-free Ringer's; 3) 0.3 mM furosemide-treated corneas; and 4) deepithelialized corneas. The swelling did not occur in corneas with silicone oil replacing the endothelium to block fluid uptake. The effluent aqueous bathing fluid from edematous corneas did not induce edema in normoxic corneas. These studies demonstrate that contact lens-induced edema depends on metabolism, involves a significant stromal contribution, and requires fluid absorption across the endothelial layer, but is not a direct result of epithelial and endothelial ion transport inhibition.

Protoporphyrin IX, a naturally occurring precursor of hemoglobin found in serum and erythrocytes, is a photoactive compound that we have detected in aqueous aspirates of three patients with hyphemas (0.13, 0.33, and 0.67 microgram/dL). To determine whether corneal endothelial photosensitization could occur, rabbit corneas were exposed to physiologic concentrations of protoporphyrin and light (200 microEinsteins [microE]/m2 per second). Experimental corneas demonstrated endothelial swelling with loss of intercellular contact by scanning electron microscopy. Flux chamber experiments demonstrated an increase in the net endothelial inflow, indicating a loss of the endothelial barrier function. Control corneas had normal ultrastructural architecture and flux study results. These studies suggest that patients with long-standing hyphemas who may be exposed to long periods of light are possibly at risk for developing endothelial dysfunction and corneal blood staining. Patching of these patients' affected eyes may be prudent.

Use of the relief mode of specular microscopy to differentiate corneal endothelial changes in anterior segment disease from retrocorneal deposits facilitates understanding of the nature of the endothelial changes. The retrocorneal deposits may be pigment granules or inflammatory deposits. The inflammatory deposits may be due to fibrin or WBCs, are more irregular than pigment, and may coalesce to form large, rounded, keratic precipitates. Pigment granules are smaller, denser, usually more regular and, although sometimes confluent, do not form keratic precipitates. Both are distinct from subendothelial blebs, both the smaller blebs up to one cell in diameter and larger blebs of two to three cells in diameter. These blebs are probably due to edema, disappear quickly on resolution of the pathologic process, and are evidence of stress to the endothelium, which shows significant change in morphology and fall in cell numbers when the blebs are of long-standing.

Two clinical cases of hematic infiltration of the cornea after severe ocular contusions are reported. Lesion of the cornea endothelium determines infiltration of the parenchyma with hemosiderin and hypofucsin, the products of hemoglobin break down. Metabolic activity of the cornea suffers, oxygen supply becomes insufficient and produces degradation of the cornea parenchyma with loss of the visual function. The affection is interpreted as a hemosiderosis process.

Contact lenses produce transient and permanent effects on the corneal endothelium in relationship to the lens oxygen transmissibility and how the contact lenses are worn. There is concern, as well as growing evidence, that contact lens-induced corneal endothelial pleomorphism causes a reduction in the endothelial functional capacity. This, in turn, may place an eye at risk for uncontrolled corneal swelling if the eye is under sufficient environmental or surgical stress. Use of lens materials and designs which promote high oxygen transmissibility, and patient wear of lenses during waking hours only, are ways to slow the development of permanent corneal endothelial pleomorphism.