A digoxin derivative that potently reduces intraocular pressure: efficacy and mechanism of action in different animal models.

Glaucoma is a blinding disease. Reduction of intraocular pressure (IOP) is the mainstay of treatment, but current drugs show side effects or become progressively ineffective, highlighting the need for novel compounds. We have synthesized a family of perhydro-1,4-oxazepine derivatives of digoxin, the selective inhibitor of Na,K-ATPase. The cyclobutyl derivative (DcB) displays strong selectivity for the human α2 isoform and potently reduces IOP in rabbits. These observations appeared consistent with a hypothesis that in ciliary epithelium DcB inhibits the α2 isoform of Na,K-ATPase, which is expressed strongly in nonpigmented cells, reducing aqueous humor (AH) inflow. This paper extends assessment of efficacy and mechanism of action of DcB using an ocular hypertensive nonhuman primate model (OHT-NHP) (Macaca fascicularis). In OHT-NHP, DcB potently lowers IOP, in both acute (24 h) and extended (7-10 days) settings, accompanied by increased aqueous humor flow rate (AFR). By contrast, ocular normotensive animals (ONT-NHP) are poorly responsive to DcB, if at all. The mechanism of action of DcB has been analyzed using isolated porcine ciliary epithelium and perfused enucleated eyes to study AH inflow and AH outflow facility, respectively. 1) DcB significantly stimulates AH inflow although prior addition of 8-Br-cAMP, which raises AH inflow, precludes additional effects of DcB. 2) DcB significantly increases AH outflow facility via the trabecular meshwork (TM). Taken together, the data indicate that the original hypothesis on the mechanism of action must be revised. In the OHT-NHP, and presumably other species, DcB lowers IOP by increasing AH outflow facility rather than by decreasing AH inflow.NEW & NOTEWORTHY When applied topically, a cyclobutyl derivative of digoxin (DcB) potently reduces intraocular pressure in an ocular hypertensive nonhuman primate model (Macaca fascicularis), associated with increased aqueous humor (AH) flow rate (AFR). The mechanism of action of DcB involves increased AH outflow facility as detected in enucleated perfused porcine eyes and, in parallel, increased (AH) inflow as detected in isolated porcine ciliary epithelium. DcB might have potential as a drug for the treatment of open-angle human glaucoma.

In-vivo imaging for assessing tumor growth in mouse models of ocular melanoma.

Uveal melanoma (UM) and conjunctival melanoma (CM) are ocular malignancies that give rise to life-threatening metastases. Although local disease can often be treated successfully, it is often associated with significant vision impairment and treatments are often not effective against metastatic disease. Novel treatment modalities that preserve vision may enable elimination of small tumors and may prevent subsequent metastatic spread. Very few mouse models of metastatic CM and UM are available for research and for development of novel therapies. One of the challenges is to follow tumor growth in-vivo and to determine the right size for treatment, mainly of the posterior, choroidal melanoma. Hence, the purpose of this study was to establish a simple, noninvasive imaging tool that will simplify visualization and tumor follow-up in mouse models of CM and UM. Tumors were induced by inoculation of murine B16LS9 cells into the sub-conjunctival or the choroidal space of a C57BL/6 mouse eye under a surgical microscope. Five to ten days following injection, tumor size was assessed by Phoenix MicronIV™ image-guided Optical Coherence Tomography (OCT) imaging, which included a real-time camera view and OCT scan of the conjunctiva and the retina. In addition, tumor size was evaluated by ultrasound and histopathological examination of eye sections. Tumor growth was observed 5-9 days following sub-conjunctival or sub-retinal injection of seven-thousand or seventy-thousand cells, respectively. A clear tumor mass was detected at these regions using the MicronIV™ imaging system camera and OCT scans. Histology of eye sections confirmed the presence of tumor tissue. OCT allowed an accurate measurement of tumor size in the UM model and a qualitative assessment of tumor size in the CM model. Moreover, OCT enabled assessing the success rate of the choroidal tumor induction and importantly, predicted final tumor size already on the day of cell inoculation. In conclusion, by using a simple, non-invasive imaging tool, we were able to follow intraocular tumor growth of both CM and UM, and to define, already at the time of cell inoculation, a grading scale to evaluate tumor size. This tool may be utilized for evaluation of new mouse models for CM and UM, as well as for testing new therapies for these diseases.

The effect of estrogen and progesterone on porcine corneal biomechanical properties.

PURPOSE: To determine the effect of the hormones estrogen and progesterone on the biomechanical properties of porcine corneas. METHODS: Thirty fresh porcine corneas were acquired from an abattoir. The corneas were equally divided into three groups. Groups were incubated for 1 week in Eusol-C solution containing supra-physiologic concentrations of estrogen, progesterone, or control (no added hormone). After incubation, the central corneal thickness (CCT) of each cornea was measured using an electronic caliper, and then the corneas were cut into strips. The strips were then clamped in the pneumatic jaws of a computer-controlled biomaterial tester (Instron 4502, USA) and stretched at a constant rate of 1 mm/min until tissue rupture while constantly recording the stress and strain of the tissue. Stress-strain curves were plotted and Young's modulus was calculated for each corneal strip. RESULTS: Average corneal thickness was 873.5 ± 143.1 µm for the control group, 928.0 ± 97.7 µm for the estrogen group, and 922.0 ± 116.7 µm for the progesterone group (data presented as mean ± SD). There was no statistically significant difference between the groups regarding the CCT (p = 0.89). The average Young's modulus was 17.00 ± 3.46 MPa for the control group, 16.95 ± 6.83 MPa for the progesterone group, and 12.33 ± 3.24 MPa for the estrogen group. The difference between the control and estrogen groups was statistically significant (p = 0.018) while the difference between the control and progesterone groups was not (p = 0.72). CONCLUSION: Estrogen has a relaxing effect on the porcine cornea, resulting in reduced stiffness of the tissue. Progesterone has no significant effect on the biomechanical properties of porcine corneas. Estrogen and progesterone do not significantly affect CCT.

Digoxin derivatives with selectivity for the α2ß3 isoform of Na,K-ATPase potently reduce intraocular pressure.

The ciliary epithelium in the eye consists of pigmented epithelial cells that express the α1ß1 isoform of Na,K-ATPase and nonpigmented epithelial cells that express mainly the α2ß3 isoform. In principle, a Na,K-ATPase inhibitor with selectivity for α2ß3 that penetrates the cornea could effectively reduce intraocular pressure, with minimal systemic or local toxicity. We have recently synthesized perhydro-1,4-oxazepine derivatives of digoxin by NaIO4 oxidation of the third digitoxose and reductive amination with various R-NH2 substituents and identified derivatives with significant selectivity for human α2ß1 over α1ß1 (up to 7.5-fold). When applied topically, the most α2-selective derivatives effectively prevented or reversed pharmacologically raised intraocular pressure in rabbits. A recent structure of Na,K-ATPase, with bound digoxin, shows the third digitoxose approaching one residue in the ß1 subunit, Gln84, suggesting a role for ß in digoxin binding. Gln84 in ß1 is replaced by Val88 in ß3. Assuming that alkyl substituents might interact with ß3Val88, we synthesized perhydro-1,4-oxazepine derivatives of digoxin with diverse alkyl substituents. The methylcyclopropyl and cyclobutyl derivatives are strongly selective for α2ß3 over α1ß1 (22-33-fold respectively), as determined either with purified human isoform proteins or intact bovine nonpigmented epithelium cells. When applied topically on rabbit eyes, these derivatives potently reduce both pharmacologically raised and basal intraocular pressure. The cyclobutyl derivative is more efficient than Latanoprost, the most widely used glaucoma drug. Thus, the conclusion is that α2ß3-selective digoxin derivatives effectively penetrate the cornea and inhibit the Na,K-ATPase, hence reducing aqueous humor production. The new digoxin derivatives may have potential for glaucoma drug therapy.

Digoxin derivatives with enhanced selectivity for the α2 isoform of Na,K-ATPase: effects on intraocular pressure in rabbits.

In the ciliary epithelium of the eye, the pigmented cells express the α1ß1 isoform of Na,K-ATPase, whereas the non-pigmented cells express mainly the α2ß3 isoform of Na,K-ATPase. In principle, a Na,K-ATPase inhibitor with selectivity for α2 could effectively reduce intraocular pressure with only minimal local and systemic toxicity. Such an inhibitor could be applied topically provided it was sufficiently permeable via the cornea. Previous experiments with recombinant human α1ß1, α2ß1, and α3ß1 isoforms showed that the classical cardiac glycoside, digoxin, is partially α2-selective and also that the trisdigitoxose moiety is responsible for isoform selectivity. This led to a prediction that modification of the third digitoxose might increase α2 selectivity. A series of perhydro-1,4-oxazepine derivatives of digoxin have been synthesized by periodate oxidation and reductive amination using a variety of R-NH2 substituents. Several derivatives show enhanced selectivity for α2 over α1, close to 8-fold in the best case. Effects of topically applied cardiac glycosides on intraocular pressure in rabbits have been assessed by their ability to either prevent or reverse acute intraocular pressure increases induced by 4-aminopyridine or a selective agonist of the A3 adenosine receptor. Two relatively α2-selective digoxin derivatives efficiently normalize the ocular hypertension, by comparison with digoxin, digoxigenin, or ouabain. This observation is consistent with a major role of α2 in aqueous humor production and suggests that, potentially, α2-selective digoxin derivatives could be of interest as novel drugs for control of intraocular pressure.

Intracameral Injection in Rats with Low Risk of Adverse Effects.

Intracameral injection is a standard administration routine in ophthalmology. The application of intracameral injection in rodents for research is challenging due to the limiting dimensions and anatomy of the eye, including the small aqueous humor volume, the lens curvature, and lens thickness. Potential damage during intracameral injections introduces adverse effects and experimental variability. This protocol describes a procedure for intracameral injection in rats, allowing precision and reproducibility. Sprague-Dawley rats were used as experimental models. Since the lens position in rats protrudes into the anterior chamber, injecting from the periphery, as done in humans, is unfavorable. Therefore, an incision is created in the central corneal region using a 31 gauge 0.8 mm stiletto blade to form a self-sealing tunnel into the anterior chamber. An incision at an angle close to the flat allows to create a long tunnel, which minimizes the loss of aqueous humor and shallowing of the anterior chamber. A 34 gauge nanoneedle is inserted into the tunnel for injection. This enables penetration with minimal friction resistance and avoids touching the lens. Injection of trypan-blue allows visualization by slit microscopy the presence of the dye in the anterior chamber and exclude leakage. Bioavailability to the corneal endothelial layer is demonstrated by injection of Hoechst dye, which stained the nuclei of corneal endothelial cells after injection. In conclusion, this protocol implements a procedure for accurate intracameral injection in rats. This procedure may be used for intracameral delivery of various drugs and compounds in experimental rat models, increasing the efficiency and reproducibility of ophthalmic research.

Mitochondria Transplantation Promotes Corneal Epithelial Wound Healing.

Purpose: The integrity of the corneal epithelium is essential in maintaining normal corneal function. Conditions disrupting the corneal epithelial layer range from chemical burns to dry eye disease and may result in impairment of both corneal transparency and sensation. Identifying factors that regulate corneal wound healing is key for the development of new treatment strategies. Here, we investigated a direct role of mitochondria in corneal wound healing via mitochondria transplantation. Methods: Human corneal epithelial cells (hCECs) were isolated from human corneas and incubated with mitochondria which were isolated from human ARPE-19 cells. We determined the effect of mitochondria transplantation on wound healing and proliferation of hCECs. In vivo, we used a mouse model of corneal chemical injury. Mitochondria were isolated from mouse livers and topically applied to the ocular surface following injury. We evaluated the time of wound repair, corneal re-epithelization, and stromal abnormalities. Results: Mitochondria transplantation induced the proliferation and wound healing of primary hCECs. Further, mitochondria transplantation promoted wound healing in vivo. Specifically, mice receiving mitochondria recovered twice as fast as control mice following corneal injury, presenting both enhanced and improved repair. Corneas treated with mitochondria demonstrated the re-epithelization of the wound area to a multi-layer appearance, compared to thinning and complete loss of the epithelium in control mice. Mitochondria transplantation also prevented the thickening and disorganization of the corneal stromal lamella, restoring normal corneal dehydration. Conclusions: Mitochondria promote corneal re-epithelization and wound healing. Augmentation of mitochondria levels via mitochondria transplantation may serve as an effective treatment for inducing the rapid repair of corneal epithelial defects.

Comparison of flanged polypropylene scleral intraocular lens fixation with scleral sutured fixation.

OBJECTIVE: To compare the outcome of 2 intraocular lens (IOL) scleral fixation techniques: double-flanged polypropylene and Hoffman scleral pocket. METHODS: Retrospective case series of all patients who underwent IOL scleral fixation by either the flange (flange group) or Hoffman scleral pocket (Hoffman group) techniques at the Kaplan Medical Center and the Edith Wolfson Medical Center. RESULTS: A total of 140 patients were included (63 flange, 77 Hoffman). The final distance-corrected visual acuity was similar between the flange and Hoffman groups (0.42 ± 0.5 and 0.51 ± 0.5 logMAR, respectively; p = 0.23), but the spherical equivalent was less myopic in the flange group (-0.63 ± 2 and -2.3 ± 1.3 D, respectively; p = 0.003). In the flange group, there were more cases of elevated IOP (17.5% vs 5.2%; p = 0.02), corneal edema (11.1% vs 1.3%; p = 0.02), cystoid macular edema (15.9% vs 2.6%; p = 0.005), and IOL decentration (19% vs 7.8%; p = 0.07). The flange group had a higher rate of combined additional procedures during the fixation surgery (68.3% vs 32%; p < 0.001), but surgery duration was not prolonged (70 vs 77 minutes; p = 0.29). CONCLUSION: Comparison of scleral IOL fixations performed with the recently developed flange technique to the conventional Hoffman scleral pocket technique resulted in similar visual outcomes and less myopization. There were more complications in the newly adopted flange technique, which may be related to the higher rate of combined anterior vitrectomy and pars plana vitrectomy. The flange technique is effective, with a shorter learning curve and similar surgical time. Therefore, it can become a viable method for scleral IOL fixation in the absence of zonular support.

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography.

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, difficulties in observing posterior choroidal melanoma in vivo limit tumor location and growth evaluation in real-time. The approach described here optimizes techniques for establishing choroidal melanoma in mice via a multi-step sub-choroidal B16LS9 cell injection procedure. To enable precision in injecting into the small dimensions of the mouse uvea, the complete procedure is performed under a microscope. First, a conjunctival peritomy is formed in the dorsal-temporal area of the eye. Then, a tract into the sub-choroidal space is created by inserting a needle through the exposed sclera. This is followed by the insertion of a blunt needle into the tract and the injection of melanoma cells into the choroid. Immediately after injection, noninvasive optical coherence tomography (OCT) imaging is utilized to determine tumor location and progress. Retinal detachment is evaluated as a predictor of tumor site and size. The presented method enables the reproducible induction of choroid-localized melanoma in mice and the live imaging of tumor growth evaluation. As such, it provides a valuable tool for studying intraocular tumors.

Oxidative stress facilitates exogenous mitochondria internalization and survival in retinal ganglion precursor-like cells.

Ocular cells are highly dependent on mitochondrial function due to their high demand of energy supply and their constant exposure to oxidative stress. Indeed, mitochondrial dysfunction is highly implicated in various acute, chronic, and genetic disorders of the visual system. It has recently been shown that mitochondrial transplantation (MitoPlant) temporarily protects retinal ganglion cells (RGCs) from cell death during ocular ischemia. Here, we characterized MitoPlant dynamics in retinal ganglion precursor-like cells, in steady state and under oxidative stress. We developed a new method for detection of transplanted mitochondria using qPCR, based on a difference in the mtDNA sequence of C57BL/6 and BALB/c mouse strains. Using this approach, we show internalization of exogenous mitochondria already three hours after transplantation, and a decline in mitochondrial content after twenty four hours. Interestingly, exposure of target cells to moderate oxidative stress prior to MitoPlant dramatically enhanced mitochondrial uptake and extended the survival of mitochondria in recipient cells by more than three fold. Understanding the factors that regulate the exogenous mitochondrial uptake and their survival may promote the application of MitoPlant for treatment of chronic and genetic mitochondrial diseases.

Excimer laser-assisted corneal epithelial pattern ablation for corneal cross-linking.

PURPOSE: To determine corneal cross-linking (CXL) efficacy and chromophore penetration after excimer laser-assisted patterned de-epithelialization. METHODS: Two-hundred-twenty porcine eyes were de-epithelialized ex vivo, either fully (mechanical; n = 88) or patterned (excimer laser; n = 132). Consecutively, corneas were impregnated with hypo- or hyperosmolar riboflavin (RF; n = 20, RF-D; n = 40, respectively) or water-soluble taurine (WST11; n = 40, and WST-D; n = 40, respectively), or kept unimpregnated (n = 80). Sixty corneas were subsequently irradiated, inducing CXL, with paired contralateral eyes serving as controls. Outcome measurements included strip extensiometry to assess CXL efficacy, and spectrophotometry and fluorescence microscopy to determine stromal chromophore penetration. RESULTS: All tested chromophores induced significant CXL (p < 0.001), ranging from 7.6% to 14.6%, with similar stiffening for all formulations (p = 0.60) and both de-epithelialization methods (p = 0.56). Light transmittance was significantly lower (p < 0.001) after full compared with patterned de-epithelialization. Stromal chromophore penetration was comparable between fully and patterned de-epithelialized samples, with full penetration in RD and RF-D samples and penetration depths measuring 591.7 ± 42.8 µm and 592.9 ± 63.5 µm for WST11 (p = 0.963) and 504.2 ± 43.2 µm and 488.8 ± 93.1 µm for WST-D (p = 0.669), respectively. CONCLUSIONS: Excimer laser-assisted patterned de-epithelialization allows for effective CXL. Stromal chromophore concentration is, however, reduced, which may have safety implications given the need for sufficient UVA attenuation in RF/UVA CXL. The different safety profile of near-infrared (NIR) may allow safe WST11/NIR CXL even with reduced stromal chromophore concentration values. In vivo studies are needed to evaluate the benefits and further assess safety of excimer laser-assisted patterned de-epithelialization for corneal CXL.

Implantation of an Artificial Endothelial Layer for Treatment of Chronic Corneal Edema.

PURPOSE: The purpose of this study was to describe a novel device that may serve as an alternative to Descemet membrane endothelial keratoplasty (DMEK) for the treatment of chronic corneal edema. METHODS: The EndoArt (EyeYon Medical, Israel) is a flexible, 50-µm thin artificial endothelial layer that matches the cornea's posterior curvature and functions as a fluid barrier at the posterior stroma, replacing the diseased endothelium. Similar to a DMEK approach, it is implanted into the anterior chamber, carefully positioned on the posterior stroma, and secured using an air-gas mixture. Two patients with chronic corneal edema resulting from endothelial decompensation underwent implantation of the new artificial lamella. RESULTS: In patient 1, the central corneal thickness (CCT) decreased from 730 µm preoperatively to 593 µm at 1 day postoperatively. In patient 2, the CCT decreased from 761 µm preoperatively to 487 µm at 1 day postoperatively. Both patients reported high satisfaction and an overall brighter visual quality. Although dislocation of the lamella occurred in both cases, the CCT was promptly restored after a repositioning procedure and remained stable at the 17-month follow-up (CCT of 526 and 457 µm for patients 1 and 2, respectively). In contrast to DMEK donor tissue, the artificial lamella is sufficiently robust to allow easy intraocular manipulation without the risk of damaging the implant. It is easily removable and does not require any immunosuppressive treatment because of its nonbiological nature. CONCLUSIONS: Implantation of the EndoArt led to rapid corneal deturgescence and CCT restoration, presenting a possible option for patients with chronic corneal edema.

Decreased Riboflavin Impregnation Time Does Not Increase the Risk for Endothelial Phototoxicity During Corneal Cross-Linking.

Purpose: To evaluate the riboflavin (RF) concentration and distribution in the corneal stroma and the risk for endothelial photodamage during corneal crosslinking (CXL) following 10- and 30-minute impregnation. Methods: De-epithelialized rabbit corneas were subjected to impregnation for 10 and 30 minutes with different RF formulations. Human corneal endothelial cells (HCECs) were subjected to different RF concentrations and ultraviolet A (UVA) dosages. Assays included fluorescence imaging, absorption spectroscopy of corneal buttons and anterior chamber humor, and cell viability staining. Results: After 10 and 30 minutes of impregnation, respectively, anterior chamber fluid showed an RF concentration of (1.6 ± 0.21)•10-4% and (5.4 ± 0.21)•10-4%, and trans-corneal absorption reported an average corneal RF concentration of 0.0266% and 0.0345%. This results in a decrease in endothelial RF concentration from 0.019% to 0.0056%, whereas endothelial UVA irradiance increases by 1.3-fold when changing from 30 to 10 minutes of impregnation. HCEC viability in cultures exposed to UVA illumination and RF concentrations as concluded for the endothelium after 10- and 30-minute impregnation was nonstatistically different at 51.0% ± 3.9 and 41.3 ± 5.0%, respectively. Conclusions: The risk for endothelial damage in CXL by RF/UVA treatment does not increase by shortened impregnation because the 30% increase in light intensity is accompanied by a 3.4-fold decrease of the RF concentration in the posterior stroma. This is substantiated by similar endothelial cell toxicity seen in vitro, which in fact appears to favor 10-minute impregnation. Translational Relevance: This study offers compelling arguments for (safely) shortening RF impregnation duration, reducing patients' burden and costly operation room time.

Enzymatic Digestion of Porcine Corneas Cross-linked by Hypo- and Hyperosmolar Formulations of Riboflavin/ultraviolet A or WST11/Near-Infrared Light.

Purpose: To assess enzymatic digestion rate after Riboflavin (RF) and Water-Soluble-Taurine (WST11) based corneal cross-linking (CXL), with or without the addition of high molecular weight dextran (RF-D and WST-D). Methods: Eighty-eight paired porcine corneas were cross-linked by either RF (n = 11) or RF-D (n = 11) and ultraviolet light (UVA), or WST11 (n = 11) or WST-D (n = 11) and near-infrared (NIR) light, or used as paired control (n = 44). Corneal buttons of treated and paired control eyes were placed in a 0.3% collagenase solution. Time to full digestion and remaining dry sample weight after six hours were compared. Results: A strong treatment effect was seen with all four formulations, as all controls had been fully digested whilst all treated samples were still visible at the experiment's endpoint. After irradiation, central corneal thickness was significantly higher in samples treated with hypo-osmolar formulations, compared to dextran enriched formulations (P < 0.001). Dry sample weight after digestion was nonsignificantly different between corneas treated by the four different formulations (P = 0.102). Average dry sample weight was 1.68 ± 0.6 (n = 10), 2.19 ± 0.50 (n = 8), 1.48 ± 0.76 (n = 11), and 1.54 ± 0.60 (n = 9) mg, for RF, RF-D, WST11, and WST-D treated samples, respectively. Enzymatic resistance was similar for RF and WST based CXL (P = 0.61) and was not affected by the addition of dextran (P = 0.221). Conclusions: Both RF and WST11 based CXL significantly increases resistance to enzymatic digestion, with similar effect for hypo-osmolar and hyperosmolar (dextran enriched) formulations. Translational Relevance: Our findings indicate these formulations are interchangeable, paving the way for the development of novel PACK-CXL protocols for thin corneas and deep-seated infections.

Therapeutic HL-Contact Lens versus Standard Bandage Contact Lens for Corneal Edema: A Prospective, Multicenter, Randomized, Crossover Study.

INTRODUCTION: To compare the safety and efficacy of the Therapeutic Hyper-CL™ lens versus a standard bandage contact lens (PureVision B&L) for chronic corneal edema. METHODS: Prospective, multicenter, randomized, crossover study. Chronic corneal edema patients were randomized to one of two arms. The first arm was fitted with the Therapeutic Hyper-CL™ lens while the second arm was fitted with a standard soft bandage contact lens. Both arms were treated with 5% sodium chloride 6 times a day. After a 7-day treatment period, there was a 7-day washout period, after which the arms were crossed over. Patients were evaluated at days 0 (baseline), 7 (following first treatment allocation), 14 (following washout), and 21 (following second treatment allocation). The primary outcomes were 3 lines of BCVA (best corrected visual acuity) improvement. RESULTS: In total, 49 patients were enrolled. There was significantly greater BCVA improvement rate >3 lines (30.4% versus 17.4%, P=0.04) in the Therapeutic Hyper-CL™ lens group. The mean change in BCVA lines was significantly greater for the Therapeutic Hyper-CL™ lens (3.4 ± 6.7 versus 0.9 ± 2.3, P=0.02). CONCLUSIONS: The Therapeutic Hyper-CL™ lens was associated with a higher chance for significant visual acuity improvement when compared to a standard bandage contact lens combined with 5% sodium chloride. This trial is registered with NCT02660151.

Repetitive magnetic stimulation protects corneal epithelium in a rabbit model of short-term exposure keratopathy.

PURPOSE: To investigate the effect of repetitive magnetic stimulation (RMS) on corneal epithelial permeability in a rabbit model of exposure keratopathy. METHODS: 61 female New Zealand White (NZW) rabbits were treated on one eye with repetitive magnetic stimulation (RMS) at a frequency of 20 Hz for 15 min. The other eye was untreated. Rabbit eyes were kept open for 2 h to induce acute corneal desiccation. The extent of fluorescein corneal staining was evaluated using EpiView software and the concentration of fluorescein in the anterior chamber was determined by a fluorometer. Safety was evaluated by electroretinogram, spectral domain optical coherence tomography (SD-OCT) and histopathology. Expression pattern of corneal cell markers was determined by immunofluorescence. RESULTS: A significant decrease in fluorescein concentration in the anterior chamber (54 ±â€¯8.4 ng/ml vs. 146.5 ±â€¯18.6 ng/ml, p = 0.000001) and in corneal surface fluorescein staining score (1.7 ±â€¯0.2 vs. 4.6 ±â€¯0.6, p = 0.00001) was obtained in RMS-treated eyes compared with control eyes, respectively. RMS treatment reduced by nearly 4 fold the percentage of corneal area with epithelial erosions by anterior segment SD-OCT. The therapeutic effect was maintained for at least 3 months. Increased expression of epithelial tight junction protein Zo-1 was observed in treated eyes. SD-OCT and histopathology analysis revealed no pathological changes in the treated or non-treated eyes. CONCLUSIONS: RMS treatment decreases epithelial corneal erosions in a rabbit model of exposure keratopathy, with no indication of pathological changes. RMS may present a novel treatment for protection of corneal epithelium from desiccation.

Multimodal Assessment of Corneal Erosions Using Optical Coherence Tomography and Automated Grading of Fluorescein Staining in a Rabbit Dry Eye Model.

PURPOSE: To evaluate the potential use of anterior segment spectral domain optical coherence tomography (AS-SD-OCT) combined with an automated grading of fluorescein staining for assessment of corneal erosions in a rabbit short-term dry eye model. METHODS: Twenty-one New Zealand white rabbits were anesthetized and eyes were kept open for 140 minutes to induce acute corneal desiccation. Rectangular scans of the cornea were performed using Spectralis AS-SD-OCT. Total corneal thickness, corneal epithelial thickness, and the percentage of epithelial erosion area (PEEA) were evaluated. Corneas were stained with fluorescein and graded automatically using EpiView and semi-automatically using ImageJ. Spearman's rank-order correlations were calculated to compare the AS-SD-OCT PEEA and the two corneal staining scores. RESULTS: Eye desiccation resulted in corneal epithelium erosions that covered 0.67% to 14.2% of the central cornea (mean ± SD: 3.95% ± 3.2%) by AS-SD-OCT. The percentage of corneal area positively stained with fluorescein ranged from 0.24% to 38.01% (mean ± SD: 12.24% ± 9.7%) by using ImageJ, correlating with the AS-SD-OCT PEEA (Spearman's ρ, 0.574; P = 0.007). The EpiView score ranged from 0.5 to 10.17 and was better correlated with the AS-SD-OCT PEEA score (Spearman's ρ, 0.795; P = 0.000017). CONCLUSIONS: Our study suggests that multimodal analysis of AS-SD-OCT and grading of fluorescein staining using EpiView software may enable quantitative assessment of corneal epithelial erosions in a rabbit short-term dry eye model. TRANSLATIONAL RELEVANCE: This multimodal imaging analysis may be applied for evaluation of superficial punctate keratitis associated with dry eye.

Homologous scleral graft for corneal perforation in a child.

PURPOSE: To show the emergency use of preserved homologous sclera for the repair of a perforated cornea in a young child. METHODS: Our case was a 3.5-year-old boy who presented with an acute corneal ulcer and endophthalmitis, which was complicated by a developing corneal melting and perforation of 2.5-mm diameter during pars plana vitrectomy. Because no donor cornea was available, a full-thickness preserved donor sclera was used to close the corneal defect. RESULTS: During the next 8 months, scar tissue formed underneath the scleral graft. The graft was removed, revealing mild opacification at the site of corneal perforation. Three years after surgery, best-corrected visual acuity was 20/60. CONCLUSIONS: In the absence of a corneal button for grafting, preserved homologous sclera may be used for closing a corneal defect.

Opacification of hydrophilic intraocular lenses associated with vitrectomy and injection of intraocular gas.

OBJECTIVE: To report 11 cases of intraocular lens (IOL) opacification after pars plana vitrectomy (PPV) involving intravitreal gas injection. METHODS AND ANALYSIS: Eleven cases of hydrophilic IOLs that opacified following PPV with intravitreal gas injection are described. Eight IOLs were explanted and analysed by light microscopy and scanning electron microscopy. Staining with alizarin red and von Kossa stains, as well as energy dispersive X-ray spectroscopy (EDX) were performed. Three IOLs were not explanted. The surgeons attached the clinical data. RESULTS: The IOLs were hydrophilic acrylic produced by six manufacturers. Six patients underwent primarily phacoemulsification with IOL implantation. PPV with intravitreal gas injection was performed 3 months-6 years afterwards. The other five patients underwent combined phacoemulsification with IOL implantation and PPV with intravitreal gas injection. IOL opacification was recorded 1 month -6 years after PPV. In eight patients, the IOLs were explanted 1 month-9 years after opacification was noticed. In three patients, the opacified IOL was not explanted. IOLs had opacified mainly anteriorly at the pupillary entrance or capsulorhexis opening. Light microscopy demonstrated granular surface deposits on the IOLs that stained positive for calcium by alizarin red and von Kossa stains. EDX analysis of the deposits detected calcium and phosphorus. CONCLUSIONS: Hydrophilic acrylic IOLs can opacify due to calcium deposition after PPV and intravitreal gas injection and may require IOL explantation. A hydrophobic IOL may be preferred when a simultaneous phacoemulsification and vitrectomy with intravitreal gas is performed.

Long-Term Biomechanical and Histologic Results of WST-D/NIR Corneal Stiffening in Rabbits, Up to 8 Months Follow-up.

Purpose: To determine the long-term safety and efficacy of WST-D/near-infrared (NIR) corneal stiffening. Methods: One eye of 23 New Zealand White rabbits was de-epithelialized mechanically followed by topical application of 2.5 mg/mL WST11, combined with dextran-500 (WST-D) for 20 minutes. Subsequently, samples were irradiated with a NIR (755 nm) laser at 10 mW/cm2 for 30 minutes. Untreated fellow eyes served as controls. One week (n = 4), 1 month (n = 6), 4 months (n = 9), or 8 months (n = 4) after treatment rabbits were euthanized. Corneal strips were cut in superior-inferior direction for extensiometry testing (1, 4, and 8 months), and histologic sections were prepared for evaluation of keratocyte distribution (1 week and 8 months). Results: Elastic modulus after treatment was significantly higher than in paired controls (16.0 ± 2.3 MPa versus 9.6 ± 3.6 MPa [P = 0.008], 18.1 ± 4.5 MPa versus 12.6 ± 2.3 MPa [P = 0.003], and 18.6 ± 3.6 MPa versus 14.2 ± 3.6 MPa [P = 0.010], at 1, 4, and 8 months, respectively). A significant decrease in keratocyte count at the anterior stroma was observed directly after treatment (1.5 ± 1.7 vs. 19.0 ± 4.1 [P = 0.002]). At 8 months keratocyte repopulation appeared completed, with similar distribution in treated and untreated corneas (15.9 ± 1.1 vs. 14.5 ± 2.5 [P = 0.562]). Corneal thickness was comparable between treated and untreated corneas at all time points. Conclusions: WST-D/NIR treatment resulted in significant and persistent long-term increase in corneal stiffness. Initial keratocyte apoptosis in the anterior stroma is followed by repopulation to normal level at 8 months after treatment. The safe nature of NIR light allows treatment of corneas of any thickness without endangering corneal endothelium or deeper ocular structures, potentially benefiting patients deemed unsuitable for riboflavin/UV-A cross-linking.