Enhanced Migration of Fuchs Corneal Endothelial Cells by Rho Kinase Inhibition: A Novel Ex Vivo Descemet's Stripping Only Model.

Descemet's Stripping Only (DSO) is a surgical technique that utilizes the peripheral corneal endothelial cell (CEnC) migration for wound closure. Ripasudil, a Rho-associated protein kinase (ROCK) inhibitor, has shown potential in DSO treatment; however, its mechanism in promoting CEnC migration remains unclear. We observed that ripasudil-treated immortalized normal and Fuchs endothelial corneal dystrophy (FECD) cells exhibited significantly enhanced migration and wound healing, particularly effective in FECD cells. Ripasudil upregulated mRNA expression of Snail Family Transcriptional Repressor (SNAI1/2) and Vimentin (VIM) while decreasing Cadherin (CDH1), indicating endothelial-to-mesenchymal transition (EMT) activation. Ripasudil activated Rac1, driving the actin-related protein complex (ARPC2) to the leading edge, facilitating enhanced migration. Ex vivo studies on cadaveric and FECD Descemet's membrane (DM) showed increased migration and proliferation of CEnCs after ripasudil treatment. An ex vivo DSO model demonstrated enhanced migration from the DM to the stroma with ripasudil. Coating small incision lenticule extraction (SMILE) tissues with an FNC coating mix and treating the cells in conjunction with ripasudil further improved migration and resulted in a monolayer formation, as detected by the ZO-1 junctional marker, thereby leading to the reduction in EMT. In conclusion, ripasudil effectively enhanced cellular migration, particularly in a novel ex vivo DSO model, when the stromal microenvironment was modulated. This suggests ripasudil as a promising adjuvant for DSO treatment, highlighting its potential clinical significance.

Ubiquinol Supplementation of Donor Tissue Enhances Corneal Endothelial Cell Mitochondrial Respiration.

PURPOSE: To determine whether ubiquinol improves mitochondrial function and cell viability in human donor corneal endothelial cells during hypothermic corneal tissue storage. METHODS: Endothelial cell Descemet membrane tissues were treated with 10 µM ubiquinol, the reduced form of the antioxidant coenzyme Q10, for 5 days in Optisol-GS storage media before assaying for mitochondrial activity using extracellular flux analysis of oxygen consumption. In addition, endothelial cell Descemet membrane tissues were analyzed for cell viability using apoptosis and necrosis assays. Control tissues from mate corneas were treated with diluent only, and comparisons were analyzed for differences. RESULTS: A total of 13 donor corneal tissues with a mean (SEM) preservation time of 11.8 days (0.4) were included for the analysis. Treatment with 10 µM ubiquinol increased spare respiratory capacity by 174% (P = 0.001), maximal respiration by 93% (P = 0.003), and proton leak by 80% (P = 0.047) compared with controls. Cells treated with ubiquinol had no significant change in cell necrosis or apoptosis. CONCLUSIONS: Preliminary testing in donor corneal tissue at specified doses indicates that ubiquinol may be a useful biocompatible additive to hypothermic corneal storage media that increases corneal endothelial cell mitochondrial function. Additional investigations are indicated to further study and optimize the dose and formulation of ubiquinol for use in preserving donor corneal tissue function during hypothermic storage.

Dietary Melatonin Therapy Alleviates the Lamina Cribrosa Damages in Patients with Mild Cognitive Impairments: A Double-Blinded, Randomized Controlled Study.

BACKGROUND Alzheimer's disease (AD) is a degenerative disease that is characterized by massive neuron devastations in the hippocampus and cortex. Mild cognitive impairment (MCI) is the transitory stage between normality and AD dementia. This study aimed to investigate the melatonin induced effects on the lamina cribrosa thickness (LCT) of patients with MCI. MATERIAL AND METHODS The LCT data of patients with MCI were compared to LCT data of healthy controls. Subsequently, all MCI patients were randomly assigned into an experimental group (with melatonin treatment) or a placebo group (without any melatonin treatment). RESULTS The LCT of MCI patients decreased significantly compared with healthy controls. The univariate analysis showed that the lower the Mini Mental State Examination (MMSE) score (P=0.038; 95% CI: 0.876, -0.209), the smaller hippocampus volume (P=0.001; 95% CI: -1.594, -2.911), and the upregulated level of cerebrospinal fluid (CSF) T-tau (P=0.036; 95% CI: 2.546, -0.271) were associated significantly with the thinner LCT in MCI patients. There were 40 patients in the experimental group and 39 patients in the placebo group. The mean age of the experimental group was not significantly different from the placebo group (66.3±8.8 versus 66.5±8.3; P>0.05). The LCT and hippocampus volume of the melatonin treated group were significantly larger compared with the placebo group (P<0.001). On the other hand, the CSF T-tau level of the melatonin treated group was significantly lower compared with the untreated group (P<0.001). CONCLUSIONS LCT assessment might allow early diagnosis of MCI. Dietary melatonin therapy could provide an effective medication for MCI patients with LCT alterations.

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes.

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet's membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

Tractional Descemet's membrane detachment after ocular alkali burns: case reports and review of literature.

BACKGROUND: Descemet's membrane detachment (DMD) is a rare complication after ocular chemical injury and its pathogenesis remains unclear. In this study, we reported two cases of DMD with traction demonstrated on Anterior segment optical coherence tomography (AS-OCT). CASE PRESENTATION: Two patients sustained ocular chemical injury with 50% sodium hydroxide. In both cases, AS-OCT revealed detached Descemet's membrane that was adherent to the underlying iris tissue in the inferior quadrant at 45 days and 34 days after the injury respectively. The first case received intracameral tamponade with 12% C3F8 gas and the second case received corticosteroid and sodium chloride 5% eye drops. However, DMD persisted in both cases. CONCLUSIONS: The atypical features of DMD on anterior segment optical coherence tomography in our cases suggested the presence of an inflammatory component caused adhesions and traction of iris to Descemet's membrane and prevented reattachment of DMD even with gas tamponade.

Effect of vital dyes on human corneal endothelium and elasticity of Descemet's membrane.

The purpose of this study was to evaluate the effects of vital dyes on human Descemet's membranes (DMs) and endothelia. DMs of 25 human cadaveric corneas with research consent were treated with dyes routinely used in Descemet membrane endothelial keratoplasty (DMEK), 0.05% Trypan blue (TB) or a combination of 0.15% Trypan blue, 0.025% Brilliant blue and 4% Polyethylene glycol (commercial name Membrane Blue Dual; MB). The effects of these two dyes on (i) endothelial cell viability, (ii) DM mechanical properties as assessed by atomic force microscopy, and iii) qualitative DM dye retention were tested for two varying exposure times (one or four minutes). No significant differences in cell toxicity were observed between treatments with TB and MB at the two different exposure times (P = 0.21). Further, both dyes led to a significant increase in DM stiffness: exposure to TB and MB for one minute increased the apparent elastic modulus of the DM by 11.2% (P = 8*10-3) and 17.7%, respectively (P = 4*10-6). A four-minute exposure led to an increase of 8.6% for TB (P = 0.004) and 13.6% for MB (P = 0.03). Finally, at 25 minutes, the dye retention of the DM was considerably better for MB compared to TB. Taken together, a one-minute exposure to MB was found to improve DM visibility compared to TB, with a significant increase in DM stiffness and without detrimental effects on endothelial cell viability. The use of MB could therefore improve (i) visibility of the DM scroll, and (ii) intraoperative unfolding, enhancing the probability of successful DMEK surgery.

Lutein and Brilliant Blue-Based Dye for Donor Preparation and Transplantation in Descemet Membrane Endothelial Keratoplasty.

PURPOSE: Trypan blue (TB) is used for visualization of the endothelium-Descemet membrane graft during Descemet membrane endothelial keratoplasty (DMEK). However, TB is assumed to have a dose-dependent toxic effect on the corneal endothelium. We retrospectively compared a lutein/zeaxanthin and Brilliant Blue (LZ/BB)-based dye for preparation and transplantation of the graft in DMEK to TB. METHODS: In 20 patients, a dye composed of 1% LZ /0.025% BB was used, and in 22 patients, 0.06% TB was used for graft visualization in DMEK. We evaluated the rebubbling rate, best spectacle-corrected visual acuity (BSCVA), central corneal thickness (CCT), and endothelial cell density (ECD) before and 3 and 6 months after transplantation. RESULTS: Staining of endothelium-Descemet membrane with LZ/BB was faint but sufficient. There was no significant difference between both groups, except in CCT after 3 months, which was lower in the LZ/BB group (P = 0.02). In the LZ/BB group, BSCVA improved from 0.48 ± 0.39 before DMEK to 0.19 ± 0.09 after 6 months (P < 0.05) (20% rebubbling rate). Donor ECD decreased from 2735 ± 259 cells/m preoperatively to 1876 ± 316 cells/mm (P < 0.0001) at 6 months (31.4%). CCT decreased from 642 ± 130 µm preoperatively to 519.8 ± 38 µm (P < 0.0001) at 6 months. In the TB group, BSCVA improved from 0.43 ± 0.27 to 0.17 ± 0.14 (P = 0.0003) at 6 months (30% rebubbling rate). ECD decreased from 2825 ± 263 to 1777 ± 302 cells/mm (P < 0.0001) after 6 months (36.3%), CCT from 638 ± 48 µm preoperatively to 531 ± 48 µm (P < 0.0001) at 6 months. CONCLUSIONS: LZ/BB-based dyes are suitable for DMEK with outcomes comparable to TB. However, available formulas result in faint staining, discouraging their routine use in donors with assumed difficult preparation.

Role of dextran in maintaining adhesive and stiffness properties of prestripped DMEK lenticules.

PURPOSE: To investigate the adhesive and stiffness properties of prestripped Descemet membrane endothelial keratoplasty (DMEK) lenticules in different preservation conditions (with and without dextran). METHODS: The study included 3 conditions: (C1) tissues collected from tissue culture media (TCM), stripped and preserved in TCM; (C2) tissues collected from transport media (TM) (TCM supplemented with 6% dextran T-500), stripped and preserved in TM; and (C3) tissues collected from TCM, stripped and preserved in TM. Using a hinge, 9.5-mm stripped DMEK lenticules were restored back on the stroma and preserved for 4 days at room temperature (RT) in different conditions as above. Nine tissues, 3 from each condition, were used to check the adhesive (fibronectin, laminin, and vitronectin) and elastic properties (fibrillin, elastin, and collagen VI) using different antibodies. Six tissues, 2 from each condition, were used to check the stiffness properties after preservation using atomic force microscopy (AFM) nanoindentation method. RESULTS: On the Descemet membrane, fibronectin was strongly expressed in C2 and C3, whereas laminin was intense in C2 postpreservation. Vitronectin was expressed in all the conditions. Elastic proteins were not expressed in either of the conditions apart from collagen VI, which was expressed on the posterior stroma. Atomic force microscopy showed higher stiffness in C3 and an insignificant but lower rigidity in C2 as compared to C1. CONCLUSIONS: The tissues from C2 showed expression of adherent proteins and lower stiffness. Dextran may be suitable in preservation of DMEK grafts before and after preparation. Less stiff tissues may help reduce manipulations required in the recipient eye during DMEK surgery.

Effect of Trypan Blue on Descemet Membrane Elasticity.

PURPOSE: To evaluate the effect of trypan blue on the elastic property of Descemet membrane (DM) by atomic force microscopy. METHODS: Human corneas (n = 10) were obtained from the Illinois Eye Bank (Chicago, IL). The DM was isolated and divided into two halves, one half was stained with ophthalmic trypan blue (Vision Blue, 0.06%, DORC International), whereas the unstained other half served as control. The DM samples were then tested using the atomic force microscope. Data were analyzed using the Hertz model for the evaluation of the Young modulus of elasticity. RESULTS: Atomic force microscopy showed higher cantilever deflection on trypan blue-stained DM compared with control, and the difference was statistically significant (P = 0.03). Force-distance curve analysis also revealed a statistically significant increase in the Young modulus of elasticity in the trypan blue-stained samples (10.5 ± 1.4 kPa) compared with the control (5.8 ± 0.8 kPa), (P < 0.0001). CONCLUSIONS: Our results suggest that trypan blue may decrease DM elasticity and consequently increase its stiffness. This may influence the graft adherence when used for endothelial keratoplasty.

Reattachment of Descemet's membrane using C3F8 gas in an eye with a Baerveldt aqueous shunt.

Descemetʼs membrane detachment (DMD) is a rare complication following cataract and glaucoma surgery as well as lamellar graft procedures. DMD can lead to blurry vision, halos and severe visual loss. Clinically, when there is a large central detachment, a double anterior chamber is seen to form. In this scenario, surgical repair may be needed. Repair of localised DMD may be achieved by injection of gases such as perfluoropropane (C3F8) and sulfurhexafluoride (SF6) or sterile air. The effect of a functioning Baerveldt tube in situ during these injections has not been reported. We report a case of DMD repair in an eye with a Baerveldt aqueous shunt.

Management of detached graft donor by SF6 injection following Descemet stripping automated endothelial keratoplasty of an eye with iridocorneal endothelial syndrome and Ahmed glaucoma drainage tube.

A 65-year-old woman with iridocorneal endothelial syndrome and a history of Ahmed glaucoma drainage (AGD) tube implantation underwent Descemet stripping automated endothelial keratoplasty (DSAEK) in her right eye. During the procedure, filling the anterior chamber with air was quite difficult due to escape of air via the AGD tube and a complete air fill of the anterior chamber could only be managed after multiple attempts. On operation night, there was no air left in the anterior chamber. On postoperative day 1, graft detachment was determined by slit-lamp biomicroscopy. For rebubbling, sulfur hexafluoride (SF(6)) 20 % was injected into the anterior chamber. Two days later, there was still some SF(6) in the anterior chamber and the graft was completely attached. At postoperative week 2, visual acuity was 2/10. SF(6) use may be considered for DSAEK in cases of previous AGD tube implantation history due to its potential for longer duration in order to obtain a better tamponade with the bubble due to its expanding nature.

Spontaneous resolution of extensive descemet membrane detachment caused by sodium cyanide injury to the eye.

PURPOSE: To describe the clinical course, ultrasonographic and confocal microscopic findings in a patient who developed a large Descemet membrane (DM) detachment that resolved spontaneously after ocular sodium cyanide injury. METHODS: Case report. RESULTS: The patient presented with severe corneal stroma edema, a large detachment of DM, iritis, and anterior subcapsular lens opacity in the left eye caused by contact with 5% sodium cyanide. The ultrasound biomicroscopic and in vivo confocal microscopic examination exhibited the configuration of the DM detachment and extensive damage of corneal cells. Although no viable endothelial cells were observed with in vivo confocal microscopy, the DM spontaneously reattached 11 days after injury, coincident with increased intraocular pressure. However, persistent corneal edema and iris atrophy were observed 6 months after DM reattached. CONCLUSIONS: Sodium cyanide can cause severe injuries to the cornea and the anterior segment. Extensive DM detachment may resolve spontaneously despite the absence of viable endothelial cells.

Effect of fibrin glue on the biomechanical properties of human Descemet's membrane.

BACKGROUND: Corneal transplantation has rapidly evolved from full-thickness penetrating keratoplasty (PK) to selective tissue corneal transplantation, where only the diseased portions of the patient's corneal tissue are replaced with healthy donor tissue. Descemet's membrane endothelial keratoplasty (DMEK) performed in patients with corneal endothelial dysfunction is one such example where only a single layer of endothelial cells with its basement membrane (10-15 µm in thickness), Descemet's membrane (DM) is replaced. It is challenging to replace this membrane due to its intrinsic property to roll in an aqueous environment. The main objective of this study was to determine the effects of fibrin glue (FG) on the biomechanical properties of DM using atomic force microscopy (AFM) and relates these properties to membrane folding propensity. METHODOLOGY/PRINCIPAL FINDINGS: Fibrin glue was sprayed using the EasySpray applicator system, and the biomechanical properties of human DM were determined by AFM. We studied the changes in the "rolling up" tendency of DM by examining the changes in the elasticity and flexural rigidity after the application of FG. Surface topography was assessed using scanning electron microscopy (SEM) and AFM imaging. Treatment with FG not only stabilized and stiffened DM but also led to a significant increase in hysteresis of the glue-treated membrane. In addition, flexural or bending rigidity values also increased in FG-treated membranes. CONCLUSIONS/SIGNIFICANCE: Our results suggest that fibrin glue provides rigidity to the DM/endothelial cell complex that may aid in subsequent manipulation by maintaining tissue integrity.

Facilitated diffusion of VEGF165 through descemet's membrane with sucrose octasulfate.

Vascular endothelial growth factor A (VEGF-A) is a promoter of neovascularization and thus a popular therapeutic target for diseases involving excessive growth of blood vessels. In this study, we explored the potential of the disaccharide sucrose octasulfate (SOS) to alter VEGF165 diffusion through Descemet's membrane. Descemet's membranes were isolated from bovine eyes and used as a barrier between two chambers of a diffusion apparatus to measure VEGF transport. Diffusion studies revealed a dramatic increase in VEGF165 transport in the presence of SOS, with little diffusion of VEGF165 across the membrane over a 10-h time course in the absence of SOS. Diffusion studies with VEGF121, a non-heparin binding variant of VEGF, showed robust diffusion with or without SOS. To determine a possible mechanism, we measured the ability of SOS to inhibit VEGF interactions with extracellular matrix (ECM), using cell-free and cell surface binding assays. Binding studies showed SOS had no effect on VEGF165 binding to either heparin-coated plates or endothelial cell surfaces at less than mg/ml concentrations. In contrast, we show that SOS inhibited VEGF165 binding to fibronectin in a dose dependent manner and dramatically accelerated the rate of release of VEGF165 from fibronectin. SOS also inhibited the binding of VEGF165 to fibronectin-rich ECM deposited by vascular smooth muscle cells. These results suggest that fibronectin-rich extracellular matrices serve as barriers to VEGF165 diffusion by providing a network of binding sites that can trap and sequester the protein. Since the content of Descemet's membrane is typical of many basement membranes it is possible that they serve throughout the body as formidable barriers to VEGF165 diffusion and tightly regulate its bioavailability and distribution within tissues.

An enzymatic technique to facilitate air separation of the stroma-Descemet's membrane junction.

PURPOSE: To describe an enzymatic technique that facilitates air separation of Descemet's membrane from the corneal stroma. METHODS: Fresh human corneoscleral tissue was mounted on an artificial anterior chamber. In a control group, air was injected into the stroma. A second group received a stromal injection of 2.5 mg/mL collagenase type 2 in balanced salt solution that was left in the stroma for 1 hour and 15 minutes. A third group received an injection of 2.5 mg/mL collagenase type 2 in balanced salt solution followed 1 hour and 15 minutes later by an injection of air into the stroma. All injections were performed with a 27-gauge needle into the deep stroma without penetrating Descemet's membrane. Anterior segment optical coherence tomography (AS-OCT), histologic examination, and electron microscopy of the junction between the stroma and Descemet's membrane were performed. The trypan blue exclusion and TUNEL assays were used to study endothelial cell viability after collagenase incubation. RESULTS: Injection of air or collagenase into the deep corneal stroma did not result in a reproducible separation of the stroma-Descemet's junction. In contrast, the stroma was easily and reproducibly separated from Descemet's membrane with a combination of intrastromal collagenase and air injection. The separation was confirmed by using light and electron microscopy. The cleavage plane seemed to be located between the junction of the posterior stroma and the anterior banded layer of Descemet's membrane. Trypan blue staining demonstrated the viability of endothelial cells after collagenase incubation. TUNEL assay confirmed excellent viability after collagenase+air separation. CONCLUSIONS: This technique facilitates the separation of Descemet's membrane from the stroma without affecting endothelial cell viability.

Presumed corneal copper deposition and oral contraceptive use.

PURPOSE: To report a case of presumed bilateral corneal copper deposition secondary to oral contraceptive use. METHODS: A 23-year-old woman was referred for evaluation of bilateral corneal opacities. The location of the deposits deep in Descemet's membrane and appearance made copper deposition a likely consideration. RESULTS: Subsequent laboratory results revealed an elevated serum copper level (189 µg/dL). Other causes of cupremia were subsequently ruled out, and the patient's corneal copper deposition was attributed to her oral contraceptive use. With cessation of the oral contraceptive, her serum copper levels normalized, but the corneal deposits remained after 5 months of follow up. CONCLUSION: To our knowledge, we report the first case in the ophthalmic literature of presumed corneal copper deposition in the setting of oral contraceptive use. It is important to recognize the corneal findings associated with copper deposition, because it may lead to the diagnosis and treatment of other serious systemic conditions causing elevated serum copper levels.