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.

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.

Evaluation of posterior capsule opacification of the Alcon Clareon IOL vs the Alcon Acrysof IOL using a human capsular bag model.

BACKGROUND: Posterior capsule opacification (PCO) after cataract surgery is influenced by intraocular lens (IOL) design and material. The following is an ex vivo comparison of PCO between the Clareon vs. the AcrySof IOL in human capsular bags. METHODS: Twenty cadaver capsular bags from 10 human donors were used, with the novel hydrophobic IOL (Clareon, CNA0T0) being implanted in one eye and the other eye of the same donor receiving the AcrySof IOL (SN60WF) following phacoemulsification cataract surgery. Five capsular bags of 3 donors served as controls without IOL. Cellular growth of lens epithelial cells was photo-documented daily. The primary endpoint was the time until full coverage of the posterior capsule by cells. Furthermore, immunofluorescence staining of capsular bags for the fibrotic markers f-actin, fibronectin, alpha smooth muscle actin, and collagen type 1 were performed. RESULTS: The new Clareon IOL did not show any disadvantages in terms of days until full cell coverage of the posterior capsule in comparison to the AcrySof (p > 0.99). Both, the Clareon (p = 0.01, 14.8 days) and the AcrySof IOL (p = 0.005, 15.7 days) showed a slower PCO development in comparison to the control (8.6 days). The fibrotic markers f-actin, fibronectin, alpha smooth muscle actin, and collagen type 1 were equally distributed between the two IOLs and differed from the control. CONCLUSIONS: A comparable performance has been found in the ex vivo formation of PCO between the two IOLs. Long-term clinical studies are necessary to reach final conclusions.

Combined VEGF/PDGF inhibition using axitinib induces αSMA expression and a pro-fibrotic phenotype in human pericytes.

PURPOSE: Large trials on anti-VEGF/PDGF (vascular endothelial/platelet-derived growth factor) combination therapy have been established to improve management of neovascular activity in age-related macular degeneration. Targeting pericytes, PDGF is thought to induce vessel regression and reduce fibrovascular scarring. The fate of pericytes exposed to anti-VEGF/PDGF combination therapy is not clear. Therefore, this study was designed to study the influence of anti-VEGF/PDGF on pericyte phenotype and cellular behavior. METHODS: Human pericytes from placenta (hPC-PL) were treated with axitinib, a tyrosine kinase inhibitor targeting VEGFR1-3 and PDGFR. Toxic effects were excluded using live/dead staining. Phenotypic changes were evaluated using phalloidin staining for actin cytoskeleton and the expression of stress fibers. MRNA and protein expression levels of α-smooth muscle actin (αSMA) as a marker of proto-myofibroblastic transition were evaluated with real-time PCR and Western blotting. Influences of fibrotic cellular mechanisms were evaluated with a scratch wound migration and a collagen gel contraction assay. RESULTS: Treatment with 0.5, 1, and 2.5 µg/ml axitinib strongly induced a proto-myofibroblast-like actin cytoskeleton with a marked increase in stress fibers. Quantitative real-time PCR and Western blotting revealed these changes to be linked to dose-dependent increases in αSMA mRNA and protein expression. However, fibrotic cellular mechanisms were significantly reduced in the presence of axitinib (scratch wound closure: up to - 78.4%, collagen gel contraction: up to - 37.4%). CONCLUSIONS: Combined anti-VEGF/PDGF inhibition seems to induce a proto-myofibroblast-like phenotype in human pericytes in vitro, but reduce profibrotic cellular mechanisms due to prolonged anti-PDGF inhibition.

Premacular membranes in tissue culture.

PURPOSE: To investigate integrity and characteristics of human premacular membranes (PMM) with and without standard tissue culturing using mechanical traction. METHODS: Premacular membranes were harvested from 32 eyes of 32 patients with idiopathic macular pucker during standard vitrectomy. By flat-mount preparation with phase contrast and interference microscopy, specimens were prepared for time-lapse microscopy, immunocytochemistry, and transmission electron microscopy. Sixteen of 32 specimens were held in tissue culture with tangential traction by using entomological pins. Of these, specimens of 7 eyes were analyzed with and without tissue culturing for comparison. Primary antibodies were used for myofibroblasts, hyalocytes, macro-/microglial cells, and retinal pigment epithelial and immune cells. RESULTS: Hyalocytes, macroglia, and microglia composed the main cell composition of surgically removed PMM. Correlation of time-lapse microscopy with immunofluorescence microscopy identified fast and unidirectional moving small round cells as microglia. Slowly moving elongated large cells were characterized as alpha-smooth muscle actin (α-SMA)-positive myofibroblasts. Following tissue culturing with tangential stretch, enhanced positive immunolabelling for α-SMA and integrins-αv was seen. All other labelling results were demonstrated to be similar with pre-culture conditions. Ultrastructural analysis revealed fibroblasts, myofibroblasts, and proliferation of glial cells following tissue culture. CONCLUSION: This study demonstrates abundance of fibroblasts, myofibroblasts, and glial cells in PMM from idiopathic macular pucker following tissue culture with tangential stretch application. We found enhanced contractive properties of the cultured PPM that appear to indicate transdifferentiation of the cell composition. This in vitro model may improve understanding of pathogenesis in traction maculopathies and help to establish further anti-fibrosis treatment strategies.

Impact of Preinjection Spectral Domain Optical Coherence Tomography Findings in the Use of Intravitreal Ocriplasmin in a Clinical Setting.

PURPOSE: To investigate the impact of spectral domain optical coherence tomography (SD-OCT) morphological predictive markers on visual acuity and outcome using ocriplasmin for macular hole and vitreomacular traction syndrome. METHODS: A series of 40 patients in a retrospective study received intravitreal ocriplasmin. The primary endpoint was defined as morphological resolution of vitreomacular traction or closure of a macular hole. We analyzed the impact of pre- and postinjection SD-OCT findings on the outcome and visual acuity. RESULTS: Thirteen of the 40 patients benefited from treatment. Statistical correlation between baseline characteristics and outcome revealed that higher foveal thickness (p = 0.018) and nontractional epiretinal membranes (p = 0.05) resulted in a worse outcome. In treatment success best corrected visual acuity gained was 9 (SD 12) letters and in failure 1 (SD 9) letter. We could not observe an influence of preinjection SD-OCT findings and other factors on visual outcome. CONCLUSION: We could confirm the therapeutic effect of ocriplasmin injections. SD-OCT morphological factors that influence treatment success and visual acuity were determined.

Combined VEGF and PDGF inhibition for neovascular AMD: anti-angiogenic properties of axitinib on human endothelial cells and pericytes in vitro.

PURPOSE: Drugs currently approved for neovascular age-related macular degeneration (nAMD) offer anti-VEGF monotherapy only. Platelet-derived growth factor (PDGF) signaling is pivotal to pericyte-induced stabilization of choroidal neovascularizations (CNV), and causes partial anti-VEGF resistance. No combination therapy for VEGF and PDGF has been approved yet. Axitinib is a tyrosine kinase inhibitor interfering with VEGF and PDGF signaling, and has been approved for the treatment of renal cell carcinoma. This study evaluates anti-angiogenic properties of axitinib in an in-vitro model of choroidal neovascularizations in nAMD. METHODS: Human endothelial cells (HUVEC) and human pericytes (hPC-PL) were treated with axitinib doses ranging from 1.0 ng/ml to 10 µg/ml. Cellular viability and proliferation were assessed with a modified MTT assay. VEGF- and PDGF-stimulated migration was observed in modified Boyden chambers. Formation of capillary structures was evaluated on Cultrex basement membrane. RESULTS: Proliferation was significantly inhibited in both cell lines in a dose-dependent manner. VEGF and PDGF significantly induced, whereas simultaneous axitinib normalized cellular migration in HUVEC and pericytes. On growth-factor-reduced Cultrex, VEGF induced the formation of capillary structures, while axitinib significantly reverted this effect. Axitinib reduced the amount of vessel associated tissue on full growth factor Cultrex. All effects on both cell lines were observed in non-toxic concentrations of axitinib. CONCLUSIONS: Axitinib inhibits angiogenesis in endothelial cells and pericytes via VEGFR and PDGFR modulation in vitro. Further studies are needed to elucidate whether axitinib may also improve therapy of CNV in AMD in vivo by interference with pericyte stabilization of pathological vessels.

A cell culture technique for human epiretinal membranes to describe cell behavior and membrane contraction in vitro.

PURPOSE: To introduce a human cell culture technique for investigating in-vitro behavior of primary epiretinal cells and membrane contraction of fibrocellular tissue surgically removed from eyes with idiopathic macular pucker. METHODS: Human epiretinal membranes were harvested from ten eyes with idiopathic macular pucker during standard vitrectomy. Specimens were fixed on cell culture plastic using small entomological pins to apply horizontal stress to the tissue, and then transferred to standard cell culture conditions. Cell behavior of 400 epiretinal cells from 10 epiretinal membranes was observed in time-lapse microscopy and analyzed in terms of cell migration, cell velocity, and membrane contraction. Immunocytochemistry was performed for cell type-specific antigens. RESULTS: Cell specific differences in migration behavior were observed comprising two phenotypes: (PT1) epiretinal cells moving fast, less directly, with small round phenotype and (PT2) epiretinal cells moving slowly, directly, with elongated large phenotype. No mitosis, no outgrowth and no migration onto the plastic were seen. Horizontal contraction measurements showed variation between specimens. Masses of epiretinal cells with a myofibroblast-like phenotype expressed cytoplasmatic α-SMA stress fibers and correlated with cell behavior characteristics (PT2). Fast moving epiretinal cells (PT1) were identified as microglia by immunostaining. CONCLUSIONS: This in-vitro technique using traction application allows for culturing surgically removed epiretinal membranes from eyes with idiopathic macular pucker, demonstrating cell behavior and membrane contraction of primary human epiretinal cells. Our findings emphasize the abundance of myofibroblasts, the presence of microglia and specific differences of cell behavior in these membranes. This technique has the potential to improve the understanding of pathologies at the vitreomacular interface and might be helpful in establishing anti-fibrotic treatment strategies.

Viability of Primary Human Pigment Epithelium Cells and Muller-Glia Cells after Intravitreal Ziv-Aflibercept and Aflibercept.

PURPOSE: The aim of this study was to access the safety profiles of 2 fusion proteins with anti-vascular endothelial growth factor action (ziv-aflibercept and aflibercept) on retinal pigment epithelium cells and Muller-Glia cells in culture by assessing cell viability post drug exposure. METHODS: Primary human retinal pigment epithelium cells (pRPE) and Muller-Glia cells (Mio-M1) were exposed to the clinical standardized concentrations of ziv-aflibercept (25 mg/mL) and aflibercept (40 mg/mL). Progressively higher concentrations of NaCl (300, 500, 1,000, 1,500, 2,000, 5,000, and 10,000 mosm/kg) were also applied to cells to assess the possibility of potentiating hyperosmotic cytotoxity effect. The study was applied to measure pRPE and Mio-M1 viability by a tetrazolium dye-reduction assay (XTT). RESULTS: Cell viability of both pRPE and Mio-M1 presented no significant changes after exposure of ziv-aflibercept and aflibercept. Progressive NaCl concentrations did not significantly alter cell viability. The exposure to the negative control of 75 µL/mL of dimethyl sulfoxide showed significant reduction in cell viability. CONCLUSIONS: At clinical doses, neither ziv-aflibercept nor aflibercept caused any significant reduction in cell viability in vitro. Furthermore, injection solutions of NaCl with higher osmolality caused no significant reduction in cell viability.

EGFR inhibitor Gefitinib attenuates posterior capsule opacification in vitro and in the ex vivo human capsular bag model.

PURPOSE: Posterior capsule opacification (PCO) occurs as a common complication after cataract surgery. Gefitinib is a selective inhibitor of the epidermal growth factor receptor (EGFR) which represents a potential pharmacological target for PCO prevention. In this in vitro study, we assessed the effect and biocompatibility of Gefitinib in PCO prophylaxis. METHODS: The effect of Gefitinib on the key pathological features of PCO was assessed in vitro. We determined growth in the human capsular bag model, prepared from sixteen cadaver eyes that underwent sham cataract surgery. Furthermore, two lens epithelial cell lines, HLE-B3 and FHL-124, were used to determine concentration-based effects on cell proliferation. In addition, cell-migration, matrix-contraction, and cell spreading were investigated. To exclude toxic concentrations, Gefitinib was assessed for its biocompatibility on six different human ocular cell types from the anterior and posterior segment of the eye. RESULTS: Gefitinib significantly increased the time until confluence of the capsular bag compared to controls (p < 0.001)). In both human lens epithelial cell lines (HLE-B3 and FHL-124), proliferation decreased significantly and as equally strong after incubation with Gefitinib (p < 0.001), as did chemotactic migration (p = 0.004), matrix contraction (p = 0.001), and cell-spreading (p = 0.001). At the IC50 concentration, Gefitinib was well tolerated by six different human ocular cell types of the anterior and posterior segment. CONCLUSION: The specific EGFR inhibitor Gefitinib might become of clinical relevance in PCO prophylaxis as it attenuated cellular growth and other pathological PCO factors in the ex vivo human capsular bag model and in two human lens epithelial cell lines, while showing good biocompatibility in vitro.

Idebenone Prevents Oxidative Stress, Cell Death and Senescence of Retinal Pigment Epithelium Cells by Stabilizing BAX/Bcl-2 Ratio.

PURPOSE: Age-related macular degeneration (AMD) is one of the leading causes of blindness. Degeneration of the retinal pigment epithelium (RPE) is pathognomonic for the disease, and oxidative stress plays an important role in the pathogenesis of this disease. This study investigates potential antiapoptotic and cytoprotective effects of idebenone on cultured RPE cells (ARPE-19) under conditions of oxidative stress. METHODS: ARPE-19 cells were treated with 1-100 µM idebenone. Cell viability (MTT assay), induction of intracellular reactive oxygen species (ROS) and histone-associated DNA fragments in mono- and oligonucleosomes, expression of proapoptotic BAX and antiapoptotic Bcl-2 as well as senescence-associated ß-galactosidase (SA-ß-Gal) activity were investigated under exposure to hydrogen peroxide (H2O2). RESULTS: Idebenone concentrations from 1 to 20 µM showed no toxic effects on ARPE-19 cells. When cells were treated with H2O2, pretreatment with 5, 7.5, 10, and 20 µM idebenone led to a significant increase in the viability of ARPE-19 cells. In addition, idebenone pretreatment significantly attenuated the induction of SA-ß-Gal and intracellular ROS as well as the amount of histone-associated DNA fragments after treatment with H2O2. The reduction of proapoptotic BAX and the elevation of antiapoptotic Bcl-2 under idebenone show that this process is rather mediated by inhibiting H2O2-induced apoptosis, not necrosis. CONCLUSION: In this study, idebenone increased survival of ARPE-19 cells and reduced cell death, senescence, and oxidative stress by stabilizing the BAX/Bcl-2 ratio.

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.

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.

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.

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.

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.

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.

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.

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.