Analysis of Cryopreservation Protocols and Their Harmful Effects on the Endothelial Integrity of Human Corneas.

Corneal cryopreservation can partially solve the worldwide concern regarding donor cornea shortage for keratoplasties. In this study, human corneas were cryopreserved using two standard cryopreservation protocols that are employed in the Tissue Bank of the Teresa Herrera Hospital (Spain) to store corneas for tectonic keratoplasties (TK protocol) and aortic valves (AV protocol), and two vitrification protocols, VS55 and DP6. Endothelial viability and general corneal state were evaluated to determine the protocol that provides the best results. The potential corneal cryopreservation protocol was studied in detail taking into consideration some cryopreservation-related variables and the endothelial integrity and stroma arrangement of the resulting cryopreserved corneas. TK corneas showed mostly viable endothelial cells, while the others showed few (AV) or none (DP6 and VS55). The corneal structure was well maintained in TK and AV corneas. TK corneas showed endothelial acellular areas surrounded by injured cells and a normal-like stromal fiber arrangement. Cryoprotectant solutions of the TK protocol presented an increasing osmolality and a physiological pH value. Cooling temperature rate of TK protocol was of 1 °C/min to -40 °C and 3 °C/min to -120 °C, and almost all of dimethyl sulfoxide left the tissue after washing. Future studies should be done changing cryopreservation-related variables of the TK protocol to store corneas of optical grade.

Ascorbic acid ameliorates corneal endothelial dysfunction and enhances cell proliferation via the noncanonical GLUT1-ERK axis.

BACKGROUND: The pumping function of corneal endothelial cells (CECs) plays a pivotal role in the maintenance of corneal water homeostasis. Corneal endothelial dysfunction (CED) leads to corneal edema and opacity, but with the exception of keratoplasty, no optimal therapeutic strategies have been established for CED. In this study, we aimed to investigate the ameliorative effect of ascorbic acid (AA) on CED and the underlying mechanism of action in the corneal endothelium. METHODS: Rabbit corneal endothelial damage was induced by anterior chamber injection of benzalkonium chloride (BAK). AA was topically administered to the corneal surface, and the transparency and thickness of the cornea were assessed by external eye photography, slit-lamp photography, and ultrasonic pachymetry. To further analyze the mechanism, rabbit CECs and immortalized human CECs (B4G12 cells) were cultured. A ferric reducing/antioxidant and AA (FRASC) assay was performed to measure the AA concentration. Cell proliferation was evaluated by cell counting and bromodeoxyuridine (BrdU) labeling assays, and protein expression was examined by liquid chromatography-mass spectrometry (LC/MS) and immunoblotting. The involvement of glucose transporter 1 (GLUT1) and phospho-ERK was evaluated via GLUT1-siRNA and phospho-ERK inhibitor (PD98059) treatment. INTERPRETATION: We observed that topical AA ameliorates BAK-induced rabbit corneal endothelial damage. Furthermore, we demonstrated that AA is transported into B4G12 cells via GLUT1, and afterward, AA increases ERK phosphorylation and promotes cell proliferation. Our findings indicate that CEC proliferation stimulated via the noncanonical AA-GLUT1-ERK axis contributes to AA-enhanced healing of CED.

Toxicity of amantadine hydrochloride on cultured bovine cornea endothelial cells.

Amantadine hydrochloride (HCl) is commonly prescribed for treating influenza A virus infection and Parkinson's disease. Recently, several studies have indicated that the use of amantadine HCl is associated with corneal edema; however, the cytotoxic effect of amantadine HCl has not been investigated. In the present study, the effects of amantadine HCl on cell growth, proliferation, and apoptosis in bovine cornea endothelial cells, and in vitro endothelial permeability were examined. Results showed that lower doses of amantadine HCl do not affect cell growth (≤ 20 µΜ), whereas higher doses of amantadine HCl inhibits cell growth (≥ 50 µΜ), induces apoptosis (2000 µΜ), increases sub-G1 phase growth arrest (2000 µΜ), causes DNA damage (≥ 1000 µΜ), and induces endothelial hyperpermeability (≥ 1000 µΜ) in bovine cornea endothelial cells; additionally, we also found that amantadine HCl attenuates the proliferation (≥ 200 µΜ) and arrests cell cycle at G1 phase (≥ 200 µΜ) in bovine cornea endothelial cells. In the present study, we measured the cytotoxic doses of amantadine HCl on cornea endothelial cells, which might be applied in evaluating the association of corneal edema.

Mitochondrial ROS Induced Lysosomal Dysfunction and Autophagy Impairment in an Animal Model of Congenital Hereditary Endothelial Dystrophy.

Purpose: The Slc4a11 knock out (KO) mouse model recapitulates the human disease phenotype associated with congenital hereditary endothelial dystrophy (CHED). Increased mitochondrial reactive oxygen species (ROS) in the Slc4a11 KO mouse model is a major cause of edema and endothelial cell loss. Here, we asked if autophagy was activated by ROS in the KO mice. Methods: Immortalized cell lines and mouse corneal endothelia were used to measure autophagy and lysosome associated protein expressions using Protein Simple Wes immunoassay. Autophagy and lysosome functions were examined in wild type (WT) and KO cells as well as animals treated with the mitochondrial ROS quencher MitoQ. Results: Even though autophagy activation was evident, autophagy flux was aberrant in Slc4a11 KO cells and corneal endothelium. Expression of lysosomal proteins and lysosomal mass were decreased along with reduced nuclear translocation of lysosomal master regulator, transcription factor EB (TFEB). MitoQ reversed aberrant lysosomal functions and TFEB nuclear localization in KO cells. MitoQ injections in KO animals reduced corneal edema and decreased the rate of endothelial cell loss. Conclusions: Mitochondrial ROS disrupts TFEB signaling causing lysosomal dysfunction with impairment of autophagy in Slc4a11 KO corneal endothelium. Our study is the first to identify the presence as well as cause of lysosomal dysfunction in an animal model of CHED, and to identify a potential therapeutic approach.

Mitochondrial Targeting of the Ammonia-Sensitive Uncoupler SLC4A11 by the Chaperone-Mediated Carrier Pathway in Corneal Endothelium.

Purpose: SLC4A11, an electrogenic H+ transporter, is found in the plasma membrane and mitochondria of corneal endothelium. However, the underlying mechanism of SLC4A11 targeting to mitochondria is unknown. Methods: The presence of mitochondrial targeting sequences was examined using in silico mitochondrial proteomic analyses. Thiol crosslinked peptide binding to SLC4A11 was screened by untargeted liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis. Direct protein interactions between SLC4A11 and chaperones were examined using coimmunoprecipitation analysis and proximity ligation assay. Knockdown or pharmacologic inhibition of chaperones in human corneal endothelial cells (HCECs) or mouse corneal endothelial cells (MCECs), ex vivo kidney, or HA-SLC4A11-transfected fibroblasts was performed to investigate the functional consequences of interfering with mitochondrial SLC4A11 trafficking. Results: SLC4A11 does not contain canonical N-terminal mitochondrial targeting sequences. LC-MS/MS analysis showed that HSC70 and/or HSP90 are bound to HA-SLC4A11-transfected PS120 fibroblast whole-cell lysates or isolated mitochondria, suggesting trafficking through the chaperone-mediated carrier pathway. SLC4A11 and either HSP90 or HSC70 complexes are directly bound to the mitochondrial surface receptor, TOM70. Interference with this trafficking leads to dysfunctional mitochondrial glutamine catabolism and increased reactive oxygen species production. In addition, glutamine (Gln) use upregulated SLC4A11, HSP70, and HSP90 expression in whole-cell lysates or purified mitochondria of HCECs and HA-SLC4A11-transfected fibroblasts. Conclusions: HSP90 and HSC70 are critical in mediating mitochondrial SLC4A11 translocation in corneal endothelial cells and kidney. Gln promotes SLC4A11 import to the mitochondria, and the continuous oxidative stress derived from Gln catabolism induced HSP70 and HSP90, protecting cells against oxidative stress.

The effects of trypan blue use on the corneal endothelium during cataract surgery in patients with pseudoexfoliation syndrome (PEX).

PURPOSE: In the present clinical study, it was aimed to investigate the possible effects of Trypan blue (TB) use on the corneal endothelium during cataract surgery in eyes with pseudoexfoliation syndrome (PEX) during a three-month follow-up period using the contralateral eye control design. METHODS: This prospective, randomised controlled, individual cohort study included 92 eyes of 46 patients with bilateral PEX and cataracts. While 1% TB was applied to one eye of the patients before capsulorhexis (study group), it was not applied to the other eye (control group). Both groups were compared preoperatively and postoperatively in terms of endothelial cell density (ECD), endothelial cell loss (%), pleomorphism, polymegathism and central corneal thickness (CCT) using specular microscopy. RESULTS: Preoperative corneal ECD was measured as 2362.56 ± 253.27 in the study group, 2380.84 ± 220.54 in the control group, and 2145.58 ± 221.71 in the study group and 2184.97 ± 200.94 cells/mm2 in the control group in the postoperative 3rd-month follow-up (p = 0.71 and = 0.37, respectively). In addition, there were no significant differences between the two groups in terms of the percentage of hexagonal cells, coefficient of variation (CV), and CCT both preoperatively and postoperatively 3 months later (p = 0.78, =0.39, =0.95 preoperatively and p = 0.31, =0.26, =0.83 postoperatively, respectively). CONCLUSION: This study demonstrated that the injection of 1% TB into the anterior chamber for staining the anterior capsule during cataract surgery did not cause significant corneal endothelial changes at postoperative 3rd months, despite the increased fragility of corneal endothelial cells in patients with PEX.

Effect of Rho-Associated Kinase Inhibitor and Mesenchymal Stem Cell-Derived Conditioned Medium on Corneal Endothelial Cell Senescence and Proliferation.

This study aims to obtain sufficient corneal endothelial cells for regenerative application. We examined the combinatory effects of Rho-associated kinase (ROCK) inhibitor Y-27632 and mesenchymal stem cell-derived conditioned medium (MSC-CM) on the proliferation and senescence of rabbit corneal endothelial cells (rCECs). rCECs were cultured in a control medium, a control medium mixed with either Y-27632 or MSC-CM, and a combinatory medium containing Y-27632 and MSC-CM. Cells were analyzed for morphology, cell size, nuclei/cytoplasmic ratio, proliferation capacity and gene expression. rCECs cultured in a combinatory culture medium showed a higher passage number, cell proliferation, and low senescence. rCECs on collagen type I film showed high expression of tight junction. The cell proliferation marker Ki-67 was positively stained either in Y-27632 or MSC-CM-containing media. Genes related to cell proliferation resulted in negligible changes in MKI67, CIP2A, and PCNA in the combinatory medium, suggesting proliferative capacity was maintained. In contrast, all of these genes were significantly downregulated in the other groups. Senescence marker ß-galactosidase-positive cells significantly decreased in either MSC-CM and/or Y-27632 mixed media. Senescence-related genes downregulated LMNB1 and MAP2K6, and upregulated MMP2. Cell cycle checkpoint genes such as CDC25C, CDCA2, and CIP2A did not vary in the combinatory medium but were significantly downregulated in either ROCK inhibitor or MSC-CM alone. These results imply the synergistic effect of combinatory culture medium on corneal endothelial cell proliferation and high cell number. This study supports high potential for translation to the development of human corneal endothelial tissue regeneration.

Molecular Hydrogen Attenuated N-methyl-N-Nitrosourea Induced Corneal Endothelial Injury by Upregulating Anti-Apoptotic Pathway.

Purpose: Previous work by our group has demonstrated the value of N-methyl-N-nitrosourea (MNU)-induced corneal endothelial decompensation in animal models. The aim of this study was to investigate the effect of molecular hydrogen (H2) on MNU-induced corneal endothelial cell (CEC) injury and the underlying mechanism. Methods: MNU-induced animal models of CEC injury were washed with hydrogen-rich saline (HRS) for 14 days. Immunofluorescence staining, immunohistochemical staining, and corneal endothelial assessment were applied to determine architectural and cellular changes on the corneal endothelium following HRS treatment. MNU-induced cell models of CEC injury were co-cultured with H2. The effect of H2 was examined using morphological and functional assays. Results: It was shown that MNU could inhibit the proliferation and specific physiological functions of CECs by increasing apoptosis and decreasing the expression of ZO-1 and Na+/K+-ATPase, whereas H2 improved the proliferation and physiological function of CECs by anti-apoptosis. Cell experiments further confirmed that H2 could reverse MNU damage to CECs by decreasing oxidative stress injury, interfering with the NF-κB/NLRP3 pathway and the FOXO3a/p53/p21 pathway. Conclusions: This study suggests that topical application of H2 could protect CECs against corneal damage factors through anti-apoptotic effect, reduce the incidence and severity of corneal endothelial decompensation, and maintain corneal transparency.

Corneal endothelial cell density in patients receiving chemotherapy.

PURPOSE: This study aimed to determine if the corneal endothelium was affected by chemotherapy. METHODS: Chemotherapy patients were recruited to undergo specular microscopy before treatment and again at 1- and 2-year follow-up visits. One eye per patient, per follow-up, was selected for comparison to baseline. RESULTS: Forty-six volunteers completed baseline and at least one follow-up assessment. From 51 eyes, there was no significant change in endothelial cell density for 41 eyes assessed at one year (MD = 0.73%, 95% CI -1.33 to 2.78%) and 18 eyes at two years (MD = 0.31%, 95% CI -3.53 to 4.15%). CONCLUSION: Although other studies have shown that chemotherapy can adversely affect the corneal epithelium, this study showed no measurable change in endothelial cell density.

Tumor necrosis factor-alpha and interferon-gamma induce inflammasome-mediated corneal endothelial cell death.

PURPOSE: Chronic corneal endothelial cell (CEC) loss results in corneal edema and vision loss in conditions such as pseudophakic bullous keratopathy (PBK), Fuchs' dystrophy, and corneal graft failure. Low CEC density has been associated with an elevation of intraocular pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α and interferon (INF)-γ. These cytokines are capable of triggering pyroptosis, a programmed cell death mechanism mediated by the inflammasome, prompting the activation of the pro-inflammatory cytokine interleukin (IL)-1ß, the perpetuation of inflammation, and subsequent damage of corneal endothelial tissue. Therefore, the purpose of this study was to determine the deleterious contribution of the inflammasome and pyroptosis to CEC loss. METHODS: CECs from human donor corneas were treated ex vivo with TNF-α and IFN-γ for 48 h. Levels of caspase-1 and IL-1ß were then assayed by ELISA, and the expression of caspase-1 and gasdermin-D (GSDM-D) were confirmed by immunofluorescence. Endothelial cell damage was analyzed by a lactate dehydrogenase (LDH) release assay, and oxidative stress was determined by measuring the levels of reactive oxygen species (ROS) in the culture media. RESULTS: Inflammasome activation and oxidative stress were elevated in CECs following exposure to TNF-α and IFN-γ, which resulted in cell death by pyroptosis as determined by LDH release which was inhibited by the caspase-1 inhibitor Ac-YVAD-cmk. CONCLUSION: CEC death is induced by the pro-inflammatory cytokines TNF-α and IFN-γ, which contribute to inflammasome activation. Moreover, the inflammasome is a promising therapeutic target for the treatment of chronic CEC loss.

Mitochondrial Antioxidant SkQ1 Improves Hypothermic Preservation of the Cornea.

Diseases of the cornea are a frequent cause of blindness worldwide. Keratoplasty is an efficient method for treating severely damaged cornea. The functional competence of corneal endothelial cells is crucial for successful grafting, which requires improving the media for the hypothermic cornea preservation, as well as developing the methods for the evaluation of the corneal functional properties. The transport of water and ions by the corneal endothelium is important for the viability and optic properties of the cornea. We studied the impact of SkQ1 on the equilibrium sodium concentration in the endothelial cells after hypothermic preservation of pig cornea at 4°C for 1, 5, and 10 days in standard Eusol-C solution. The intracellular sodium concentration in the endothelial cells was assayed using the fluorescent dye Sodium Green; the images were analyzed with the custom-designed CytoDynamics computer program. The concentrations of sodium in the pig corneal endothelium significantly increased after 10 days of hypothermic preservation, while addition of 1.0 nM SkQ1 to the preservation medium decreased the equilibrium concentration of intracellular sodium (at 37°C). After 10 days of hypothermic preservation, the permeability of the plasma membrane for sodium decreased in the control cells, but not in the cells preserved in the presence of 1 nM SkQ1. Therefore, SkQ1 increased the ability of endothelial cells to restore the intracellular sodium concentration, which makes SkQ1 a promising agent for facilitating retention of the functional competence of endothelial cells during cold preservation.

Multiple roles of FGF10 in the regulation of corneal endothelial wound healing.

Corneal endothelial dysfunction usually induces corneal haze and oedema, which seriously affect visual function. The main therapeutic strategy for this condition is corneal transplantation, but the use of this strategy is limited by the shortage of healthy donor corneas. Compared with corneal transplantation, drug intervention is less invasive and more accessible; thus, finding an effective pharmaceutical alternative for cornea transplantation is critical for the treatment of corneal endothelial dysfunction. In this study, we established a rabbit scratch model to investigate the effect of fibroblast growth factor 10 (FGF10) on corneal endothelial wound healing. Results showed that FGF10 injection accelerated the recovery of corneal transparency and increased the protein expression levels of ZO1, Na+/K+-ATPase and AQP-1. Moreover, FGF10 significantly inhibited the expression levels of endothelial-to-mesenchymal transition proteins and reduced the expression levels of the proinflammatory factors IL-1ß and TNF-α in the anterior chamber aqueous humour. FGF10 also enhanced the Na+/K+-ATPase activity by enhancing mitochondrial function as a result of its direct interaction with its conjugate receptor. Thus, FGF10 could be a new pharmaceutical preparation as treatment for corneal endothelial dysfunction.

Cytomegalovirus endotheliitis with recurrent anterior uveitis and secondary glaucoma misdiagnosed as HLA-B27 uveitis.

A 61-year-old male patient presented with decreased vision and recurrent redness in his right eye since the past 4 years. He had been diagnosed elsewhere as HLA-B27 positive anterior uveitis and was on oral methotrexate and topical corticosteroids for recurrent disease. He was on maximal medical therapy for glaucoma. Examination showed prominent inferior corneal oedema with pigmented keratic precipitates and elevated intraocular pressure. He underwent combined trabeculectomy with mitomycin C and cataract surgery. The aqueous sample tested positive for cytomegalovirus. He responded well to oral valganciclovir with resolution of uveitis, the intraocular pressure was well controlled and the corneal oedema resolved completely.

The effect of intravitreal anti-vascular endothelial growth factor injections on corneal endothelium in patients with diabetic macular oedema.

PURPOSE: To evaluate the characteristics of corneal parameters in patients with diabetic macular oedema (DME) treated with intravitreal anti-vascular endothelial growth factor (anti-VEGF) injections. METHODS: Participants in this study were 36 patients with DME, treated with either intravitreal ranibizumab (n = 16) or aflibercept (n = 20). All participants underwent best-corrected visual acuity (BCVA) measurement, optical coherence tomography and non-contact specular microscopy to evaluate corneal endothelium parameters (endothelial cell density-ECD, hexagonality, coefficient of variation of the cell size and central corneal thickness-CCT), at baseline and at months 6 and 12 after the first intravitreal injection. Comparisons between baseline and months 6 and 12 were performed. RESULTS: There was no statistically significant difference regarding ECD, hexagonality, coefficient of variation of the cell size and CCT at month 6 and 12 post initial injection compared to baseline in patients with DME. BCVA improved significantly at month 6 and 12 compared to baseline (p < 0.001 for both comparisons). Central retinal thickness was significantly reduced at month 6 and 12 compared to baseline (p < 0.001 for both comparisons). CONCLUSION: Intravitreal anti-VEGF injections in patients with DME were found not to affect corneal parameters, namely ECD, hexagonality, coefficient of variation of the cell size and CCT at the long-term follow-up of 12 months.

Potential Functional Restoration of Corneal Endothelial Cells in Fuchs Endothelial Corneal Dystrophy by ROCK Inhibitor (Ripasudil).

PURPOSE: Rho-associated kinase (ROCK) inhibitors have been successfully used as a rescue strategy in eyes that failed to clear after descemetorhexis without endothelial graft for treatment of Fuchs endothelial corneal dystrophy (FECD). The functional mechanisms by which ROCK inhibitors modulate corneal endothelial cell regeneration in FECD patients have, however, not been clarified. Here, we analyzed the effect of the ROCK inhibitor ripasudil on corneal endothelial cells of FECD patients and normal donors using ex vivo tissue and in vitro cellular models. DESIGN: Experimental study: laboratory investigation. METHODS: This institutional study used endothelial cell-Descemet membrane lamellae from FECD patients (n = 450) undergoing Descemet membrane endothelial keratoplasty (FECD ex vivo model), normal research-grade donor corneas (n = 30) after scraping off central endothelial cells (ex vivo wound healing model), normal donor corneas (n = 20) without endothelial injury, and immortalized cell lines (n = 3) generated from FECD patients (FECD in vitro model). Descemet membrane lamellae were dissected into halves and incubated for 24-72 hours in storage medium with or without a single dose of 30 µM ripasudil. The effects of ripasudil on expression of genes and proteins related to endothelial cell proliferation, migration, functionality, and endothelial-to-mesenchymal transition were analyzed and complemented by functional assays on FECD cell lines. RESULTS: A single dose of ripasudil induced significant upregulation of genes and proteins related to cell cycle progression, cell-matrix adhesion and migration, as well as endothelial barrier and pump function up to 72 hours, whereas classical markers of endothelial-to-mesenchymal transition were downregulated in both FECD and normal specimens compared to unstimulated controls ex vivo. In addition to stimulation of proliferation and migration, ripasudil-induced changes in expression of functional signature genes could be also verified in FECD cell lines in vitro. CONCLUSIONS: These data support the concept that inhibition of ROCK signaling represents a potent tool in regenerative therapies in FECD patients through reactivation of cell proliferation and migration as well as restoration of endothelial pump and barrier function without inducing adverse phenotypic changes.

Optimizing Visualization of Descemet Membrane Endothelial Keratoplasty Tissue: Assessing the Impact of Trypan Blue Exposure on Stain Duration and Corneal Endothelial Cell Function.

PURPOSE: To assess how trypan blue staining affects Descemet membrane endothelial keratoplasty (DMEK) graft visibility and corneal endothelial cell (CEC) mitochondrial respiration. METHODS: DMEK grafts (n = 20) were stained with trypan blue 0.06% for 1, 3, 5, or 10 minutes. Each graft was injected into an artificial anterior chamber. Surgery was simulated with tapping and sweeping motions on the corneal surface and injections of balanced salt solution (BSS). Graft visibility was assessed at 5, 10, 20, and 30 minutes. Effects of trypan blue on mitochondrial respiration were assessed using primary CECs cultured from donor corneas (n = 43). Treatment wells exposed to trypan blue 0.06% (1, 5, or 30 minutes) and donor-matched control wells to methylene blue 1% (1 minute) or BSS (1, 5, or 30 minutes) were assayed for key respiration parameters. RESULTS: After 5 minutes of surgical manipulation, grafts stained for 5 minutes were significantly more visible than grafts stained for 1 or 3 minutes; there was no added benefit of staining for 10 minutes. After 10 minutes of surgical manipulation, grafts stained for 3 minutes were more visible than grafts stained for 1 minute, without additional benefits of staining ≥5 minutes. No visibility differences were observed after ≥20 minutes of surgical manipulation. CEC mitochondrial respiration did not change significantly following trypan blue exposure for all intervals tested compared to BSS. CONCLUSIONS: Staining DMEK grafts with trypan blue for 3 to 5 minutes optimizes visibility during surgical manipulation without mitochondrial impairment. Corneal surgeons learning DMEK will benefit from optimizing this critical step.

Morphological change and recovery of corneal endothelial cells after rho-associated protein kinase inhibitor eye-drop (ripasudil 0.4%) instillation.

BACKGROUND/AIMS: To investigate the time-dependent change of corneal endothelial cell (CEC) morphology and density (CECD) in patients with glaucoma post instillation of rho-associated protein kinase inhibitor ripasudil (Rip) 0.4% eye drops. METHODS: This observational study involved 163 eyes of 163 patients with glaucoma in whom CEC morphological change was evaluated by CECD calculated via non-contact specular microscopy (NCSM) before and at 1 or 3 months post-Rip instillation. The change of CECD was plotted along with elapsed time from last instillation of Rip. The patients were divided into the following three groups based on the elapsed time post-Rip instillation: Early Group (<2 hours), Middle Group (≥2 hours, yet <6 hours) and Late Group (≥6 hours). The rate of CECD change was then analysed and compared among the three groups. An additional eight eyes of four patients with glaucoma were enrolled for a time-dependent study, with NCSM images evaluated before and at 1, 2, 3, 4 and 6 hours post-Rip instillation. RESULTS: Morphological changes in the CECs appeared within 1 hour and recovered to normal within 6 hours post instillation. In the Early, Middle and Late Group, the median rate of CECD change as calculated by the NCSM automated software was -5.68%, -4.95% and -0.07%, respectively. The CEC images showed the same morphological changes with observational study in all four cases. CONCLUSION: Due to transient morphological changes, the NCSM software produced misleading data for determining CECD within 1 hour post-Rip instillation, yet revealed that CEC morphology gradually recovered to normal within 6 hours.

Endothelial Biopsy for the Diagnosis and Management of Culture-Negative Retrocorneal Fungal Keratitis With the Assistance of Optical Coherence Tomography Imaging.

PURPOSE: To report a case of a culture-negative deep fungal corneal infection that was diagnosed after histopathology of an anterior segment optical coherence tomography-guided endothelial biopsy. METHODS: A 22-year-old woman with history of contact lens wear and concomitant topical steroid use presented with a mid-stromal corneal infiltrate that failed to respond to oral acyclovir and topical fortified antibiotics. Although cornea stains, cultures, and confocal microscopy showed negative results, there was high clinical suspicion for fungal keratitis. After 2 months on topical natamycin, oral voriconazole, and serial intrastromal and intracameral voriconazole injections, the infiltrate enlarged and deepened. Imaging with anterior segment optical coherence tomography revealed that the infection had progressed to an endothelial plaque. RESULTS: Diagnostic endothelial biopsy was performed in the operating room. Cultures showed again negative results, whereas histopathology of the removed specimen revealed fungal elements. The postoperative edema at the site of the biopsy resolved over the course of 4 weeks, and a posterior stromal scar formed. Serial intrastromal and intracameral voriconazole injections were continued for the first postoperative month. At the 1-year and the 3-year follow-up examinations, the patient's vision was 20/20 without recurrence. CONCLUSIONS: Intraoperative scraping of the endothelial plaque and histopathologic evaluation of the specimen proved to be of utmost importance for definitive diagnosis and resolution of the culture-negative deep fungal infection in this case. This young patient's cornea was retained and vision remains excellent.

Evaluation of corneal safety in systemic lupus erythematosus patients undergoing long-term hydroxychloroquine treatment.

PURPOSE: The aim of this study was to examine the effects of long-term use of hydroxychloroquine (HQ) on the pachymetric, aberrometric, and densitometric values of the cornea and corneal endothelium in lupus patients. METHOD: Twenty-two eyes (study group) of 22 patients using HQ for treatment of lupus and 25 eyes (control group) of 25 healthy individuals were included in this prospective study. A specular microscopy was used to measure corneal endothelial cell density (ECD), percentage of hexagonal cells (HEX%), coefficient of variation of the cell size (CV). Then, a Pentacam® HR corneal tomography system was used to measure central corneal thickness (CCT), corneal aberrometry values in 6-mm pupil diameters and corneal densitometry values in 6-mm corneal zones (0-2 mm and 2-6 mm). RESULTS: While ECD was significantly lower in the study group than in the control group (p = 0.034), CCT was significantly higher in the study group (p = 0.032). The higher-order aberrations values and the anterior corneal densitometry values in the 0-2 mm and 2-6 mm corneal zones in the study group were found to be significantly higher than the control group (p = 0.021, p = 0.007 and p = 0.013). CONCLUSION: Prolonged use of HQ may cause some changes in the cornea. In the follow-up of these cases, detailed examination of the cornea as well as the macula may be important for the protection of corneal health.