Keratoconus Progression Determined at the First Visit: A Deep Learning Approach With Fusion of Imaging and Numerical Clinical Data.

Purpose: Multiple clinical visits are necessary to determine progression of keratoconus before offering corneal cross-linking. The purpose of this study was to develop a neural network that can potentially predict progression during the initial visit using tomography images and other clinical risk factors. Methods: The neural network's development depended on data from 570 keratoconus eyes. During the initial visit, numerical risk factors and posterior elevation maps from Scheimpflug imaging were collected. Increase of steepest keratometry of 1 diopter during follow-up was used as the progression criterion. The data were partitioned into training, validation, and test sets. The first two were used for training, and the latter for performance statistics. The impact of individual risk factors and images was assessed using ablation studies and class activation maps. Results: The most accurate prediction of progression during the initial visit was obtained by using a combination of MobileNet and a multilayer perceptron with an accuracy of 0.83. Using numerical risk factors alone resulted in an accuracy of 0.82. The use of only images had an accuracy of 0.77. The most influential risk factors in the ablation study were age and posterior elevation. The greatest activation in the class activation maps was seen at the highest posterior elevation where there was significant deviation from the best fit sphere. Conclusions: The neural network has exhibited good performance in predicting potential future progression during the initial visit. Translational Relevance: The developed neural network could be of clinical significance for keratoconus patients by identifying individuals at risk of progression.

Conditions for modifying intraocular lenses as drug carriers for methotrexate using poly (lactic-co-glycolic acid).

INTRODUCTION: The intraocular lens (IOL) can be used as a slow-release drug carrier in cataract surgery to alleviate posterior capsular opacification (PCO). The following is a systematic development of an IOL using methotrexate and the solvent casting process with poly (lactic-co-glycolic acid) (PLGA) as a carrier polymer. METHODS: Different solvents for PLGA and methotrexate were tested for dissolution properties and possible damage to the IOL. The required biological concentration of methotrexate was determined in human capsular bags implanted with an IOL. To detect fibrosis, α-SMA, f-actin, and fibronectin were labelled by immunofluorescence staining. Cell proliferation and extracellular matrix contraction were observed in a lens epithelial cell line (FHL-124). Finally, the IOL was designed, and an ocular pharmacokinetic model was used to measure drug release. RESULTS: Solvent mixtures were found to allow coating of the IOL with drug and PLGA without damaging it. PCO in the capsular bag model was inhibited above 1 µM methotrexate (p = 0.02). Proliferation in FHL-124 was significantly reduced above a concentration of 10 nM (p = 0.04) and matrix contraction at 100 nM (p = 0.02). The release profile showed a steady state within therapeutic range. CONCLUSION: After determination of the required physicochemical manufacturing conditions, a drug releasing IOL was designed. A favourable release profile in an ocular pharmacokinetics model could be shown.

The Use of Corneoscleral Grafts to Maintain Tectonic Stability in Severe Keratolysis.

Severe corneal ulcerations, causing major keratolysis with large perforation of the cornea or extending to the limbal region, are an ophthalmic emergency. In these cases, a larger corneoscleral graft can be transplanted to restore tectonic integrity, alleviate pain, save vision, and prevent loss of the eye. Chart review of 34 patients with a corneoscleral graft ≥9.5 mm was conducted. Primary endpoints of the study were tectonic stability defined as no need for another keratoplasty or enucleation. In addition, visual acuity, postoperative complications, and secondary procedures were analyzed. In total, 12 patients (35%) were female. The mean age at transplantation was 65 ± 19 years. The underlying disease was a perforated infectious corneal ulcer in 30 cases (88%). Mean follow up was 675 ± 789 days. Tectonic stability at the end of the follow-up was maintained with a probability of 56% in a Kaplan-Meier analysis. Another penetrating keratoplasty was necessary in six cases (17%) and enucleation in five cases (15%). A corneoscleral transplant remains a viable treatment option to prevent enucleation in severe keratolysis. In our study, this was possible in about half of the cases. Postoperative complications, secondary surgeries, and markedly reduced visual acuity put the advantages into perspective.

Successful treatment of multidrug-resistant Pseudomonas aeruginosa keratitis with meropenem eye drops - a case report.

BACKGROUND: This case report describes the course and therapeutic management of a fast-spreading bacterial keratitis caused by multidrug-resistant (MDR) Pseudomonas aeruginosa (P. aeruginosa). CASE PRESENTATION: A 27-year-old male contact lens wearer presented with a multi-resistant, fast spreading P. aeruginosa keratitis. After initial resistance to various antibiotic therapies, testing revealed a MDR P. aeruginosa. The keratitis was treated successfully with specially prepared 50 mg/ml off-label meropenem eye drops for 18 days as well as systemic meropenem for seven days with rapid improvement of the corneal infiltrate. CONCLUSION: This case report demonstrates the combination of topical and systemic meropenem as a useful treatment option for corneal ulcers caused by MDR P. aeruginosa.

Influence of endotamponade on anterior chamber depth and refractive outcome after combined phacovitrectomy: case-control study.

PURPOSE: To compare the changes in the anterior chamber depth (ACD) and in the refractive outcomes after combined phacovitrectomy with respect to the endotamponade (balanced salt solution, air, sulfur hexafluoride [SF 6 , gas]). SETTING: Department of Ophthalmology, University Hospital Ulm, Ulm, Germany. DESIGN: Retrospective, longitudinal case-control study. METHODS: 160 eyes of 160 patients were included in the study. 120 eyes underwent phacoemulsification with in-the-bag implantation combined with vitrectomy and were divided into 3 groups according to tamponade (balanced salt solution, air, gas). 40 control eyes with cataract surgery only were included. Further inclusion criteria were uneventful surgery, no postoperative complications and absence of corneal pathology. Endpoints were ACD as measured by swept-source optical coherence tomography-based biometry (IOLMaster 700) preoperatively, 1 to 2 days and 6 weeks postoperatively and refractive prediction error (PE) using the Barrett and Haigis formulas. RESULTS: Within the first 2 days after surgery the ACD was shallower in the eyes left with gas or air tamponade, when compared with balanced salt solution or cataract surgery alone ( P < .001). This effect diminished 6 weeks later, and all eyes reached comparable ACD ( P = .396). The refractive PE was slightly, but statistically significantly higher in the gas group when compared with cataract surgery alone ( P = .012 for Barrett, P = .006 for Haigis). CONCLUSIONS: The resulting ACD after combined phacovitrectomy was independent of the tamponade used, but a gas-tamponade was associated with a higher refractive PE.

The role of the calcofluor white staining in the diagnosis of Acanthamoeba keratitis.

PURPOSE: Acanthamoeba keratitis is often misdiagnosed at disease onset. This study presents data to confirm the diagnosis using calcofluor white (CFW) staining. METHODS: Forty three patients were retrospectively included who presented to the Department of Ophthalmology at the University Hospital Ulm with keratitis between 2000 and 2022. Condition positive cases were diagnosed based on the typical clinical presentation of Acanthamoeba keratitis with a positive polymerase chain reaction (PCR). Condition negative were patients with ulcers due to other causing pathogens with a negative Acanthamoeba PCR result. The condition was compared with the CFW test results. RESULTS: After symptom onset, time until presentation was 17 ± 12 days and until diagnosis 27 ± 13 days in the 15 condition positive patients. Among the 35 patients with additional CFW test, 7 patients were condition positive and 28 negative. 5 of the 7 patients were true positive, 2 were false negative. In the 28 condition negative patients, 1 was false positive. Sensitivity of CFW was 71% and specificity 96%. The positive PCR results were available 3.4 ± 2.3 days after corneal scraping, the positive CFW test results on the same day in each case. CONCLUSION: Our data demonstrate that diagnosis of Acanthamoeba keratitis remains difficult and therapy is initiated late. A positive CFW test confirms the diagnosis as there are almost no false positive results and it was available faster than PCR. In case of a negative CFW test, Acanthamoeba keratitis cannot be ruled out because of a high false negative rate.

Pharmacological Therapy of Proliferative Vitreoretinopathy: Systematic In Vitro Comparison of 36 Pharmacological Agents.

Purpose: Proliferative vitreoretinopathy (PVR) is currently treated surgically. Reliable pharmaceutical options would be desirable, and numerous drugs have been proposed. This in vitro study is intended to systematically compare and determine the most promising candidates for the treatment of PVR. Methods: A structured literature review was conducted in the "PubMed" database to identify previously published agents proposed for medical treatment of PVR -36 substances that met the inclusion criteria. Toxicity and antiproliferative effects were evaluated on primary human retinal pigment epithelial (hRPE) using colorimetric viability assays. The seven substances with the widest therapeutic range between toxicity and no longer detectable antiproliferative effect were then validated with a bromodeoxyuridine assay and a scratch wound healing assay using primary cells derived from surgically excised human PVR membranes (hPVR). Results: Among 36 substances, 12 showed no effect on hRPE at all. Seventeen substances had a significant (P < 0.05) toxic effect of which nine did not have an antiproliferative effect. Fifteen substances significantly reduced hRPE proliferation (P < 0.05). The seven most promising drugs with the highest difference between toxicity and antiproliferative effects on hRPE were dasatinib, methotrexate, resveratrol, retinoic acid, simvastatin, tacrolimus, and tranilast. Whereof resveratrol, simvastatin, and tranilast additionally showed antiproliferative and dasatinib, resveratrol, and tranilast antimigratory effects on hPVR (P < 0.05). Conclusion: This study presents a systematic comparison of drugs that have been proposed for PVR treatment in a human disease model. Dasatinib, resveratrol, simvastatin, and tranilast seem to be promising and are well-characterized in human use.

Epiretinal Amniotic Membrane in Complicated Retinal Detachment: a Clinical and In Vitro Safety Assessment.

INTRODUCTION: Amniotic membrane (AM) is a popular treatment for external ocular diseases. First intraocular implantations in other diseases reported promising results. Here, we review three cases of intravitreal epiretinal human AM (iehAM) transplantation as an adjunct treatment for complicated retinal detachment and analyze clinical safety. Possible cellular rejection reactions against the explanted iehAM were evaluated and its influence was assessed on three retinal cell lines in vitro. METHODS: Three patients with complicated retinal detachment and implanted iehAM during pars plana vitrectomy are retrospectively presented. After removal of the iehAM at subsequent surgery, tissue-specific cellular responses were studied by light microscopy and immunohistochemical staining. We investigated the influence of AM in vitro on retinal pigment epithelial cells (ARPE-19), Müller cells (Mio-M1), and differentiated retinal neuroblasts (661W) . An anti-histone DNA ELISA for cell apoptosis, a BrdU ELISA for cell proliferation, a WST-1 assay for cell viability, and a live/dead assay for cell death were performed. RESULTS: Despite the severity of the retinal detachment, stable clinical outcomes were obtained in all three cases. Immunostaining of the explanted iehAM showed no evidence of cellular immunological rejection. In vitro, there was no statistical significant change in cell death or cell viability nor were proliferative effects detected on ARPE-19, Müller cells, and retinal neuroblasts exposed to AM. CONCLUSION: iehAM was a viable adjuvant with many potential benefits for treatment of complicated retinal detachment. Our investigations could not detect any signs of rejection reactions or toxicity. Further studies are needed to evaluate this potential in more detail.

Recurrence of perforation and overall patient survival after penetrating keratoplasty versus amniotic membrane transplantation in corneal perforation.

PURPOSE: The following is a comparative analysis on the treatment outcomes of corneal perforations using amniotic membrane transplantation (AMT) or penetrating keratoplasty (PK). METHODS: This monocentric retrospective study was performed at the Department of Ophthalmology, University Hospital Ulm, Germany. A total of 78 eyes of 78 patients were included. Thirty-nine eyes received an AMT, and 39 patients were treated with a PK. Primary outcome was recurrence of perforation. Secondary outcomes were patient mortality and visual acuity. RESULTS: No statistically significant difference was observed with regard to a recurrence of perforation between the two groups (26% in AMT vs 23% in PK, p > 0.99). The time of recurrences was within the first two years and did not differ statistically (p = 0.97). In addition, a proportional hazards model with cox regression regarding recurrent perforation showed no significant differences (p = 0.5). After AMT, 41% and after KP, 28% of the patients died during follow-up (p = 0.2), respectively. The Charlson Comorbidity Index (p < 0.0001) and the age at the time of surgery (p = 0.0002) were statistically significantly higher in those who were deceased. A mean follow-up of 485 ± 517 days was recorded. CONCLUSION: Both surgical methods show good results and no statistically significant difference regarding recurrent perforation rate. About a third of the patients died during the follow-up period. The decision regarding the appropriate method should therefore be based on a combination of all factors.

Inhibition of proliferative vitreoretinopathy by a newly developed methotrexate loaded drug carrier in vitro.

PURPOSE: Repeated intravitreal injections of methotrexate for proliferative vitreoretinopathy, a rare ocular condition that can cause vision loss, have shown beneficial effects in recent clinical studies. The purpose of this study was to develop a slow-release, long-term drug carrier composed of the polymer polylactide-co-glycolide and methotrexate that can be injected intravitreally. METHODS: The required composition of the drug carrier was modeled using pharmacokinetic parameters based on current literature. Release kinetics were determined using an ocular pharmacokinetic model. Epiretinal PVR-membranes were harvested during pars plana vitrectomy and subsequently transferred to cell culture. The effect of the drug carrier on cell migration was investigated using time-lapse microscopy and a scratch-induced migration assay. The colorimetric WST-1-assay and a live-dead-assay were performed to determine viability, and the BrdU-assay was applied for proliferation. RESULTS: The release profile showed an initial and a final burst of methotrexate with an intervening steady state that lasted 9-11 weeks. It showed inhibitory effects on pathobiological processes in human PVR-cells in vitro. Cell velocity in the time-lapse assay, migration in the scratch assay (p = 0.001), and proliferation in the BrdU assay (p = 0.027) were reduced after addition of the drug carrier. These effects occurred without causing a reduction in viability in the WST-1 assay (p > 0.99) and the live-dead assay. CONCLUSION: The methotrexate-loaded drug carrier can maintain a stable concentration for 9-11 weeks and influence the pathobiological process of PVR cells in vitro. Therefore, it represents a potential therapeutic orphan drug for PVR.

Pharmacological drug screening to inhibit uveal melanoma metastatic cells either via EGF-R, MAPK, mTOR or PI3K.

AIM: To screen five potential pharmacological substances specifically targeting EGF-R, MAPK, mTOR, or PI3K for their antiproliferative effects, possible impact on cell viability, as well as cell death rates on three different uveal melanoma metastasis cell lines in vitro. METHODS: Three different uveal melanoma metastasis cell lines (OMM2.5, OMM2.3, and OMM1), that originated from human hepatic and subcutaneous metastasis, were exposed to inhibitors of different targets: erlotinib (EGF-R), everolimus (mTOR), selumetinib (MAPK), trametinib (MAPK) or the alkylphosphocholine erufosine (PI3K). Cell viability was assessed with a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) dye reduction assay after 24h of treatment. Antiproliferative effects were evaluated separately after a 72-hour incubation of the cells with the pharmacological substance. Subsequently, the IC50 was calculated. Tumor cell death was investigated using a double stain apoptosis detection assay. RESULTS: Selumetinib, trametinib, and erufosine significantly decreased cell viability of all OMM cell lines (P<0.04). In addition, selumetinib and trametinib showed a significant inhibition of cell proliferation (P<0.05). Everolimus and erlotinib solely inhibited cell proliferation at the used concentrations (P<0.05). Besides an increase of necrotic cells after erufosine treatment (P<0.001), no changes in the number of dead cells for the other substances were observed. CONCLUSION: The preliminary drug screening demonstrates five new candidates, successfully targeting the canonical MAPK/ERK and PI3K/AKT/mTOR pathways in uveal melanoma metastasis cells in vitro. Hence, these findings provide an experimental basis to explore future single or combined therapy strategies for metastatic uveal melanoma.

Galectin-1 and -3 in high amounts inhibit angiogenic properties of human retinal microvascular endothelial cells in vitro.

PURPOSE: Galectin-1 and -3 are ß-galactoside binding lectins with varying effects on angiogenesis and apoptosis. Since in retinal pigment epithelial cells high amounts of human recombinant galectin (hr-GAL)1 and 3 inhibit cell adhesion, migration and proliferation, we investigated if hr-GAL1 and 3 have homologous effects on human retinal microvascular endothelial cells (HRMEC) in vitro. METHODS: To investigate the effect of galectin-1 and -3 on HRMEC, proliferation, apoptosis and viability were analyzed after incubation with 30, 60 and 120 µg/ml hr-GAL1 or 3 by BrdU-ELISA, histone-DNA complex ELISA, live/dead staining and the WST-1 assay, respectively. Further on, a cell adhesion as well as tube formation assay were performed on galectin-treated HRMEC. Migration was investigated by the scratch migration assay and time-lapse microscopy. In addition, immunohistochemical staining on HRMEC for ß-catenin, galectin-1 and -3 were performed and ß-catenin expression was investigated by western blot analysis. RESULTS: Incubation with hr-GAL1 or 3 lead to a decrease in proliferation, migration, adhesion and tube formation of HRMEC compared to the untreated controls. No toxic effects of hr-GAL1 and 3 on HRMEC were detected. Intriguingly, after treatment of HRMEC with hr-GAL1 or 3, an activation of the proangiogenic Wnt/ß-catenin signaling pathway was observed. However, incubation of HRMEC with hr-GAL1 or 3 drew intracellular galectin-1 and -3 out of the cells, respectively. CONCLUSION: Exogenously added hr-GAL1 or 3 inhibit angiogenic properties of HRMEC in vitro, an effect that might be mediated via a loss of intracellular endogenous galectins.

Anti-angiogenic properties of rapamycin on human retinal pericytes in an in vitro model of neovascular AMD via inhibition of the mTOR pathway.

PURPOSE: Choroidal neovascularizations (CNV) are partially stabilized through a coverage of pericytes leading to a partial anti-VEGF resistence. Drugs licensed for neovascular AMD (nAMD) do not take this mechanical and growth factor-driven CNV stability into account. The purpose of this work was to see if inhibiting the mammalian target of rapamycin (mTOR) may successfully block angiogenic cellular pathways in primary human retinal pericytes in an in vitro model of nAMD. METHODS: The mTOR inhibitor rapamycin was used to treat human retinal pericytes (HRP) at doses ranging from 0.005 to 15 g/ml. A modified metabolism-based XTT-Assay was used to assess toxicity and anti-proliferative effects. A scratch wound experiment showed the effects on migration. On Cultrex basement membrane gels, the influence of rapamycin on the development of endothelial cell capillary-like structures by human umbilical vein vascular endothelial cells (HUVEC) in the absence and presence of pericytes was investigated. RESULTS: Rapamycin showed no signs of toxicity within its range of solubility. The drug showed dose dependent anti-proliferative activity and inhibited migration into the scratch wound. Endothelial cell tube formation in a HUVEC monoculture was effectively inhibited at 45%. A co-culture of HUVEC with pericytes on Cultrex induced endothelial tube stabilization but was disrupted by the addition of rapamycin leading to degradation of 94% of the tubes. CONCLUSIONS: Rapamycin allows for an efficient modulation of aspects of angiogenesis in pericytes via mTOR-modulation in vitro. Further studies are needed to elucidate whether rapamycin may have an impact on CNV in nAMD in vivo.

In vitro evaluation of simulated stereotactic radiotherapy for wet age-related macular degeneration on three different cell lines.

Low energy stereotactic radiotherapy has been proposed for the treatment of neovascular age related macular degeneration. We investigated the in vitro effect of the radiotherapy on pericytes, retinal pigment epithelium and endothelial cells. Primary human retinal pigment epithelium cells, human umbilical vein endothelial cells and human pericytes from Placenta were cultivated. In a pairwise protocol, one plate was irradiated at a dose of 16 Gy, while the second plate served as a non-irradiated control. Thereafter, cells were cultivated either in serum-free (non-permissive) or serum-stimulated (permissive) conditions. A life/dead assay, an XTT and a BrdU assay were performed up to 7 days after irradiation. No cell death occurred at any timepoint in any cell line after treatment nor in the control. Compared to the unirradiated controls, cell viability and metabolic activity were significantly reduced in irradiated cells in the XTT assay, except for non-permissive RPE cells. In the BrdU assay, proliferation was inhibited. While no cell death was detected in vitro, viability and proliferative capacity of all cell lines were significantly reduced. Therefore, it seems that low energy stereotactic radiotherapy inhibits angiogenesis without a direct induction of apoptosis but influencing microvascular function and stability.

Development of a drug-eluting intraocular lens to deliver epidermal growth factor receptor inhibitor gefitinib for posterior capsule opacification prophylaxis.

PURPOSE: Different molecular targets, such as the epidermal growth factor receptor, have been identified for the prophylaxis of posterior capsule opacification. This led to the proposal of several drugs, yet drug delivery into the capsular bag remains challenging. The intraocular lens as a drug delivery device would provide a convenient method to allow drug release in the location needed. This is to evaluate the effect of a drug-eluting intraocular lens using an epidermal growth factor receptor inhibitor. METHODS: Hydrophobic and hydrophilic intraocular lenses were coated with gefitinib using the dip coating technique. The cellular response on the modified intraocular lenses was tested in a human lens epithelial cell line (FHL-124) in an anterior segment model. Furthermore, modified intraocular lenses were implanted into human capsular bags ex vivo. Drug release was determined as well as the biocompatibility on human corneal endothelial cells. Unmodified intraocular lenses served as controls. In addition, immunofluorescence staining with fibronectin as a marker for fibrotic response was conducted. RESULTS: Both coated hydrophilic and hydrophobic intraocular lenses could attenuate the cell growth of FHL-124 cells in the human capsular bag in comparison to the unmodified controls. Furthermore, gefitinib-soaked intraocular lenses showed a constant drug release over the first 10 days. No reduction in cell viability of corneal endothelial cells occurred. A decrease in fibronectin expression under gefitinib treatment could be observed. CONCLUSION: In vitro epidermal growth factor receptor seems to be a valuable target for the prevention of posterior capsule opacification. The gefitinib-eluting intraocular lens in this study could inhibit cell growth in non-toxic concentrations.

Cytoprotective effect of crocin and trans-resveratrol on photodamaged primary human retinal pigment epithelial cells.

PURPOSE: Light-induced damage to retinal pigment epithelium during pars plana vitrectomy remains a hot topic in ophthalmology. Improvements in technology led to a change of light sources, selective filters, and shorter light exposure time. Currently, there is no satisfying solution to the problem. The aim of the study was to investigate the cytoprotective effects of crocin and resveratrol on light-induced damage to primary human retinal pigment epithelial cells in vitro. METHODS: Primary human retinal pigment epithelial cells were exposed to light analogous to the illumination during pars plana vitrectomy. To evaluate the cytoprotective effects and potential toxicity of resveratrol and crocin, human retinal pigment epithelial cells were incubated with varying concentrations of both before 3-[4,5-dimethylthiazol-2-yl] tetrazolium bromide (MTT) viability assay. Furthermore, glutathione levels were measured to investigate synergistic antioxidant potential. Apoptosis of human retinal pigment epithelial cells was determined by a nucleosome detection enzyme-linked immunosorbent assay. RESULTS: Crocin and resveratrol improved cell viability in photodamaged human retinal pigment epithelial cells significantly from 40.65 ± 21.99% in illuminated human retinal pigment epithelial cells and reached a peak viability of 85.64 ± 11.37% in crocin and resveratrol pretreated cells (for all: p < 0.001). In line, the combination of the supplements increased glutathione levels significantly from 39.35 ± 21.96% to 80.74 ± 10.32% (p = 0.017). No toxic effects were detected (p > 0.99). However, no change in apoptosis rates could be observed following pretreatment with crocin and resveratrol (p > 0.99). CONCLUSION: Crocin and trans-resveratrol revealed cytoprotective effects on human retinal pigment epithelial cells supporting both supplement's development as potential perioperative treatments in light-induced retinal pigment epithelial damage.

Establishing an objective biomarker for corneal cystinosis using a threshold-based Spectral domain optical coherence tomography imaging algorithm.

PURPOSE: The purpose of the present study was to establish a semi-automated threshold-based image segmentation algorithm to detect and objectively quantify corneal cystine crystal deposition in ocular cystinosis with anterior segment optical coherence tomography (AS-OCT). METHODS: This prospective, observational, comparative study included 88 eyes of 45 patients from the German Cystinosis Registry Study as well as 68 eyes of 35 healthy control subjects. All eyes were imaged with AS-OCT (Cirrus HD-OCT 5000, Carl Zeiss Meditec AG, Jena, Germany). As an initial step, B-scan images were subjectively analysed for typical changes in morphology in comparison to healthy controls. Based on the experience gained, an objective semi-automated B-scan image segmentation algorithm was developed using a grey scale value-based threshold method to automatically quantify corneal crystals. RESULTS: On AS-OCT B-scans, corneal crystals appeared as hyperreflective deposits within the corneal stroma. The crystals were distributed either in all stromal layers (43 eyes, 49%) or confined to the anterior (23 eyes, 26%) or posterior stroma (22 eyes, 25%), respectively. The novel automatic B-scan image segmentation algorithm was most efficient in delineating corneal crystals at higher grey scale thresholds (e.g. 226 of a maximum of 255). Significant differences in suprathreshold grey scale pixels were observable between cystinosis patients and healthy controls (p < 0.001). In addition, the algorithm was able to detect an age-dependent depth distribution profile of crystal deposition. CONCLUSION: Objective quantification of corneal cystine crystal deposition is feasible with AS-OCT and can serve as a novel biomarker for ocular disease control and topical treatment monitoring.

Corneal optical density in Fuchs endothelial dystrophy determined by anterior segment optical coherence tomography.

PURPOSE: In this study, we propose a method to grade corneal stromal opacity using optical density measurements by anterior segment optical coherence tomography (AS-OCT) and validate the approach in Fuchs endothelial corneal dystrophy (FECD). METHODS: A retrospective analysis of human corneal OCT scans was performed on 48 eyes of 32 patients with FECD and 33 control eyes of 21 patients using the Carl Zeiss Cirrus HD-OCT 5000. In addition, corneal edema in fresh rabbit cadaver eyes was artificially induced by distilled water and imaged with the Thorlabs TELESTO-II spectral domain OCT at different time points during saturation. The increase of opacity due to corneal edema was proposed to directly correlate with enhanced reflectivity sites in the OCT images, corresponding to higher optical density. The increase was determined as the image area above a statistically established gray-scale value using ImageJ and correlated with other disease characteristics. RESULTS: Optical densities in human corneas showed significant differences between FECD patients and the control group (p = 0.002). The increased optical densities determined in FECD corneas correlated well with other disease characteristics such as corneal pachymetry or visual acuity. Likewise, rabbit corneas showed a time dependent increase in thickness and in corneal optical density during soaking in distilled water. CONCLUSION: This study presents corneal optical density by AS-OCT as an objective value for corneal changes in FECD. Complementing other diagnostic tools in FECD the assessment of corneal optical density may identify progression of FECD, gauge novel therapeutic strategies and support risk and benefit analyses for corneal surgery.

Arginine as an Enhancer in Rose Bengal Photosensitized Corneal Crosslinking.

Purpose: Oxygen-independent cornea crosslinking (CXL) using rose bengal (RB) and green light may have unique clinical applications. These studies were designed to gain insight into the arginine (arg)-enhanced anaerobic crosslinking process, to maximize crosslinking efficiency, and to test a clinically feasible method for oxygen-free CXL. Methods: Rabbit corneas were treated ex vivo using 1 mM RB and 532 nm light. RB photodecomposition, monitored by absorption spectrophotometry, was used to optimize arg concentration and to develop an irradiation and re-dying protocol. The minimal effective green light fluence was identified by linear tensile strength measurements. RB penetration into the stroma was determined by fluorescence microscopy. To favor the anaerobic pathway, a contact lens was used to minimize stromal oxygen level during irradiation. Stromal cell toxicity was evaluated by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Results: RB photodecomposition reached 75% of its maximal effect at 200 mM arg and the optimal fluence increment was 32.7 J/cm2. The minimal effective fluence for cornea stiffening was 65.4 J/cm2. Placement of a contact lens promoted oxygen-independent cornea stiffening, similar to that obtained on isolated, oxygen-deprived cornea. RB penetration into the stroma with arg present was limited to ∼120 µm, about 25% deeper than without arg. Stromal cell toxicity was limited to the depth of RB and arg penetration. Conclusions: An oxygen-independent pathway in cornea for RB-CXL was characterized and optimized, including a possible clinical protocol for its use. Translational Relevance: Oxygen-independent RB-CXL is an efficient and effective process that can be developed further for unique clinical applications.

Corneal Stromal Filler Injection as a Novel Approach to Correct Presbyopia-An Ex Vivo Pilot Study.

Purpose: To evaluate the ex vivo feasibility of corneal stromal filler injection to create bifocality to correct presbyopia by flattening the central posterior corneal surface and thus increase refractive power. Methods: Femtosecond laser-assisted corneal stromal pockets of varying diameters close to the posterior corneal curvature were cut into rabbit eyes ex vivo. Subsequently, hyaluronic acid was injected to flatten the central posterior curvature. Refractive parameters were determined using perioperatively acquired three-dimensional optical coherence tomography (OCT) scans. Using micrometer-resolution OCT, corneal endothelial cell morphology and density were evaluated. Results: Following filler injection into the corneal stromal pockets, a fair volume-dependent increase of central refractive power up to 4 diopters (dpt) was observed. Unremarkable refractive changes of the peripheral posterior (3 mm, 0.20 ± 0.11 dpt; 2 mm, 0.11 ± 0.10 dpt) and the anterior corneal curvature (3 mm, 0.20 ± 0.34 dpt; 2 mm, 0.33 ± 0.31 dpt) occurred. Only negligible changes in astigmatism were observed. Different sizes of optical zones could be established. Furthermore, no alterations of corneal endothelial morphology or endothelial cell density (2831 ± 356 cells/mm2 vs. 2734 ± 292 cells/mm2; P = 0.552) due to the adjacent laser treatment were observed. Conclusions: The ex vivo investigations proved the principle of injecting a filler material into femtosecond laser-created corneal stromal pockets close to the posterior corneal curvature as an efficacious, individually adjustable, and novel approach to correct presbyopia without ablating corneal tissue. Translational Relevance: Due to the aging population worldwide, presbyopia is an increasing problem; thus, our study may encourage further exploration to extend the treatment spectrum of clinically used femtosecond laser systems to correct presbyopia.