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1.
Acta Ophthalmol ; 101(2): e215-e225, 2023 Mar.
Article in English | MEDLINE | ID: mdl-36178238

ABSTRACT

PURPOSE: Descemet membrane endothelial keratoplasty (DMEK) accounts for >50% of all corneal transplants in Germany. So far, no data from such a large multicenter study have been published. METHODS: This retrospective study included 3200 DMEKs at seven departments performed for Fuchs endothelial corneal dystrophy (FECD) or bullous keratopathy (BK). We evaluated best corrected visual acuity (BCVA, logMAR), endothelial cell density (ECD, cells/mm2 ), minimal corneal thickness (CT, µm), rebubbling-, primary transplant failure- and immune reaction-rate. Changes over time were evaluated by linear mixed models for repeated measures and correlation with case number by center by weighted linear regression. RESULTS: For patients without vision-limiting comorbidities (74% of all analysed eyes, n = 2270), mean BCVA improved from 0.6 ± 0.4 logMAR to 0.2 ± 0.2 logMAR 6 months (p < 0.001, n = 1441) and 0.1 ± 0.2 logMAR 12 months (p = 0.001, n = 1402) postoperatively. BK- had a worse BCVA compared to FECD-patients (0.3 ± 0.5 vs. 0.1 ± 0.2 logMAR [p < 0.001] at 1 year). ECD declined from 2465 ± 259 cells/mm2 (n = 2876 preoperatively) to 1587 ± 433 cells/mm2 after 12 months (p < 0.001, n = 1237). Mean rebubbling rate was 0.4 ± 0.7/eye. 784 eyes (25%) received at least one rebubbling. More rebubblings correlated with a lower ECD, a worse BCVA, a higher CT, and higher transplant failure and rejection rates (p < 0.001, p = 0.013 for BCVA at 12 months). A single rebubbling did not influence the BCVA (p = 0.785). Graft failure rate was 3% (n = 67), rejection rate 1.5% (n = 48). CONCLUSION: Descemet membrane endothelial keratoplasty increases visual acuity with low transplant failure- and rejection-rates. FECD has a better outcome than BK. Since a quarter of all patients need a rebubbling, this should be included in the informed consent. Remarkably, one rebubbling has no influence on the outcome.


Subject(s)
Descemet Stripping Endothelial Keratoplasty , Fuchs' Endothelial Dystrophy , Humans , Endothelium, Corneal/transplantation , Retrospective Studies , Descemet Stripping Endothelial Keratoplasty/methods , Cell Count , Fuchs' Endothelial Dystrophy/surgery , Descemet Membrane/surgery , Germany/epidemiology , Treatment Outcome
2.
Br J Ophthalmol ; 106(6): 831-838, 2022 06.
Article in English | MEDLINE | ID: mdl-33472769

ABSTRACT

BACKGROUND: Voretigene neparvovec is a gene therapeutic agent for treatment of retinal dystrophies caused by bi-allelic RPE65 mutations. In this study, we report on a novel and objective evaluation of a retinotopic photoreceptor rescue. METHODS: Seven eyes of five patients (14, 21, 23, 24, 36 years, 1 male, 4 females) with bi-allelic RPE65 mutations have been treated with voretigene neparvovec. The clinical examinations included visual acuity testing, dark-adapted full-field stimulus threshold (FST), dark-adapted chromatic perimeter (DAC) with a 30-degree grid, and a 30 degrees grid scotopic and photopic chromatic pupil campimetry (CPC). All evaluations and spectral domain optical coherence tomography were performed at baseline, 1 month and 3 months. RESULTS: All except the oldest patient had a measurable improvement of the rod function assessed via FST, DAC or scotopic CPC at 1 month. The visual acuity improved slightly or remained stable in all eyes. A cone function improvement as measured by photopic CPC was observed in three eyes. The gain of the dark-adapted threshold with blue FST and the DAC stimuli (cyan) average correlated strongly with age (R2>0.7). The pupil response improvement in the scotopic CPC correlated with the baseline local retinal volume (R2=0.5). CONCLUSIONS: The presented protocols allow evaluating the individual spatial and temporal effects of gene therapy effects. Additionally, we explored parameters that correlated with the success of the therapy. CPC and DAC present new and fast ways to assess functional changes in retinotopic maps of rod and cone function, measuring complementary aspects of retinal function.


Subject(s)
Retinal Dystrophies , Female , Humans , Male , Retina , Retinal Dystrophies/genetics , Tomography, Optical Coherence , Visual Acuity , Visual Field Tests
3.
Clin Exp Ophthalmol ; 48(5): 666-681, 2020 07.
Article in English | MEDLINE | ID: mdl-32077190

ABSTRACT

BACKGROUND: Hydrogen peroxide (H2 O2 ) can be used in vitro to simulate oxidative stress. In retinal organ cultures, H2 O2 induces strong neurodegeneration of the retina. It is known that oxidative stress plays a role in the development of several retinal diseases including glaucoma and ischemia. Thus, we investigated whether processes underlying oxidative stress can be prevented by hypothermia using an ex vivo organ culture model of porcine retinas. METHODS: Porcine retinal explants were cultivated for 5 and 8 days. Oxidative stress was induced via 300 µM H2 O2 on day 1 for 3 hours. Hypothermia treatment at 30°C was applied simultaneously with H2 O2 , for 3 hours. Retinal ganglion cells (RGCs), apoptosis, bipolar and cholinergic amacrine cells, microglia and macroglia were evaluated immunohistologically. Apoptosis rate was additionally analysed via western blot. RESULTS: Reduced apoptosis rates through hypothermia led to a preservation of RGCs (P < .001). Amacrine cells were rescued after hypothermia treatment (P = .17), whereas bipolar cells were only protected partly. Additionally, at 8 days, microglial response due to oxidative stress was completely counteracted via hypothermia (P < .001). CONCLUSIONS: H2 O2 induced strong degenerative processes in porcine retinas. The role of oxidative stress in the progression of retinal diseases makes this ex vivo organ culture model suitable to investigate new therapeutic approaches. In the present study, the damaging effect of H2 O2 to several retinal cell types was counteracted or strongly alleviated through hypothermia treatment. Especially RGCs, which are affected in glaucoma disease, were protected due to a reduced apoptosis rate through hypothermia.


Subject(s)
Hypothermia , Retinal Diseases , Animals , Apoptosis , Oxidative Stress , Retina , Retinal Diseases/etiology , Retinal Diseases/prevention & control , Retinal Ganglion Cells , Swine
4.
Acta Ophthalmol ; 98(2): e155-e160, 2020 Mar.
Article in English | MEDLINE | ID: mdl-31469490

ABSTRACT

PURPOSE: The purpose of this study was to establish a standardized in vitro phacoemulsification damage model for future investigations of the effects of phacoemulsification, surgical devices, protective ophthalmic viscoelastic devices (OVDs), irrigation solutions and other aspects related to cataract phacoemulsification surgery on the corneal endothelium using porcine eyes. METHODS: Thirty-four porcine eyes were randomly assigned to three groups (phacoemulsification (n = 13), irrigation (n = 9), control (n = 12)). A total of 5 min of ultrasound energy with intermittent irrigation/aspiration was applied in the eyes of the phacoemulsification group. The eyes of the irrigation group received the identical treatment, but without the application of ultrasound energy. The control group was left untreated. All eyes were then prepared to split corneal buttons followed by 15 days of cultivation. Endothelial cell density (ECD) was assessed blinded on day 15. RESULTS: Endothelial cell density declined significantly more until day 15 in the phacoemulsification group (2567 ± 317/267 cells/mm² (median ± 25%/75%-quartiles), -32.5 ± 7.0/6.4%) compared to the irrigation (3450 ± 350/383 cells/mm², -11.8 ± 5.3/2.6%; p < 0.001) and the control group (3650 ± 288/258 cells/mm², -10.2 ± 3.2/4.6%; p < 0.001). CONCLUSION: The phacoemulsification damage model presented in this study is sensitive to phacoemulsification energy and may reliably be used to investigate various factors involved in phacoemulsification with regard to their influence on corneal endothelial cells. This method is able to replace animal experiments or in vitro cell culture experiments that often do not translate well to the in vivo situation in humans.


Subject(s)
Corneal Endothelial Cell Loss/etiology , Disease Models, Animal , Endothelium, Corneal/pathology , Phacoemulsification/adverse effects , Ultrasonic Surgical Procedures/adverse effects , Animals , Cell Count , Corneal Endothelial Cell Loss/pathology , Organ Culture Techniques , Swine , Therapeutic Irrigation , Viscoelastic Substances
5.
Clin Exp Ophthalmol ; 47(8): 1043-1054, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31152487

ABSTRACT

BACKGROUND: Hypoxia contributes to retinal damage in several retinal diseases, including central retinal artery occlusion, with detrimental consequences like painless, monocular loss of vision. Currently, the treatment options are severely limited due to the short therapy window, as the neuronal cells, especially the retinal ganglion cells (RGCs), are irreversibly damaged within the first few hours. Hypothermia might be a possible treatment option or at least might increase the therapy window. METHODS: To investigate the neuroprotective effect of hypothermia after retinal hypoxia, an easy-to-use ex vivo retinal hypoxia organ culture model developed in our laboratory was used that reliably induced retinal damage on a structural, molecular and functional level. The neuroprotective effect of hypothermia after retinal hypoxia was analysed using optical coherence tomography scans, histological stainings, quantitative real-time polymerase chain reaction, western blotting and microelectrode array recordings. RESULTS: Two different hypothermic temperatures (30°C and 20°C) were evaluated, both exhibited strong neuroprotective effects. Most importantly, hypothermia increased RGC survival after retinal hypoxia. Furthermore, hypothermia counteracted the hypoxia-induced RGC death, reduced macroglia activation, attenuated retinal thinning and protected from loss of spontaneous RGC activity. CONCLUSIONS: These results indicate that already a mild reduction in temperature protects the RGCs against damage and could function as a promising therapeutic option for hypoxic diseases.


Subject(s)
Hypothermia, Induced , Hypoxia/pathology , Retina/pathology , Retinal Ganglion Cells/cytology , Animals , Apoptosis , Blotting, Western , Cell Survival/physiology , Cytoprotection , Gene Expression Regulation/physiology , Glial Fibrillary Acidic Protein/genetics , Immunohistochemistry , Microelectrodes , Organ Culture Techniques , RNA, Messenger/genetics , Rats , Real-Time Polymerase Chain Reaction , Retina/metabolism , Retinal Ganglion Cells/metabolism , Tomography, Optical Coherence
6.
Sci Rep ; 9(1): 4898, 2019 03 20.
Article in English | MEDLINE | ID: mdl-30894574

ABSTRACT

Simulation of hypoxic processes in vitro can be achieved through cobalt chloride (CoCl2), which induces strong neurodegeneration. Hypoxia plays an important role in the progression of several retinal diseases. Thus, we investigated whether hypoxia can be reduced by hypothermia. Porcine retinal explants were cultivated for four and eight days and hypoxia was mimicked by adding 300 µM CoCl2 from day one to day three. Hypothermia treatment (30 °C) was applied simultaneously. Retinal ganglion, bipolar and amacrine cells, as well as microglia were evaluated via immunohistological and western blot analysis. Furthermore, quantitative real-time PCR was performed to analyze cellular stress and apoptosis. In addition, the expression of specific marker for the previously described cell types were investigated. A reduction of ROS and stress markers HSP70, iNOS, HIF-1α was achieved via hypothermia. In accordance, an inhibition of apoptotic proteins (caspase 3, caspase 8) and the cell cycle arrest gene p21 was found in hypothermia treated retinae. Furthermore, neurons of the inner retina were protected by hypothermia. In this study, we demonstrate that hypothermia lowers hypoxic processes and cellular stress. Additionally, hypothermia inhibits apoptosis and protects neurons. Hence, this seems to be a promising treatment for retinal neurodegeneration.


Subject(s)
Amacrine Cells , Cold Temperature , Microglia , Retinal Bipolar Cells , Retinal Ganglion Cells , Amacrine Cells/metabolism , Amacrine Cells/pathology , Animals , Apoptosis , Apoptosis Regulatory Proteins/metabolism , Biomarkers/metabolism , Cell Hypoxia , Cobalt , In Vitro Techniques , Microglia/metabolism , Microglia/pathology , Neurodegenerative Diseases/pathology , Neurodegenerative Diseases/therapy , Reactive Oxygen Species/metabolism , Retinal Bipolar Cells/metabolism , Retinal Bipolar Cells/pathology , Retinal Diseases/pathology , Retinal Diseases/therapy , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/pathology , Swine
7.
Eye (Lond) ; 33(3): 358-362, 2019 03.
Article in English | MEDLINE | ID: mdl-30209266

ABSTRACT

PURPOSE: To present a new surgical technique for treating corneal opacity and aniridia with aphakia and the results in a small consecutive case series. METHODS: A three-piece acrylic intraocular lens (IOL) was attached to a customized silicone iris prosthesis and fixed with three 10-0 polypropylene sutures in a knotless technique using Z-sutures after trephination of the recipient cornea. The medical records of all consecutive patients who had received a keratoplasty and an implantation of an artificial iris and IOL were reviewed. RESULTS: Five eyes of five patients were included in the analysis. The mean age of the patients was 46.2 years and the mean follow-up was 24.6 months. The mean best-corrected visual acuity improved from 1.36 logMAR before surgery to 0.78 logMAR after surgery during the follow-up. At the last follow-up visit, the artificial iris-IOL complex was well centered with good positioning in all cases. CONCLUSIONS: Management of post-traumatic aniridia combined with aphakia and corneal scars or graft failure by haptic fixation of a foldable IOL on an artificial iris combined with a simultaneous keratoplasty appears to be a promising approach, which allows to correct a complex lesion with a less traumatic and faster procedure.


Subject(s)
Iris/surgery , Keratoplasty, Penetrating/methods , Lens Implantation, Intraocular/methods , Lenses, Intraocular , Prostheses and Implants , Adult , Follow-Up Studies , Humans , Iris/injuries , Keratoplasty, Penetrating/rehabilitation , Male , Middle Aged , Patient Satisfaction/statistics & numerical data , Suture Techniques , Treatment Outcome , Visual Acuity
8.
Acta Ophthalmol ; 96(8): e970-e973, 2018 Dec.
Article in English | MEDLINE | ID: mdl-30239156

ABSTRACT

PURPOSE: To confirm the reproducibility of manual graft preparation using curvilinear forceps and evaluate the incidence and type of structural abnormalities of Descemet's membrane (DM) preventing successful grafts preparation. METHODS: Five hundred corneo-scleral buttons were prepared. Factors such as endothelial cell number before preparation, donor age, post-mortem time, time in culture, pigmentation of the trabecular meshwork and preparation characteristics of the fellow eye were analysed. According to the preparation characteristics, three groups were formed: A, uncomplicated; B, complicated preparation with stripping from the contralateral side; and C, failure of preparation. Three failed grafts were examined by transmission electron microscopy (TEM). RESULTS: Using curvilinear forceps, manual separation of DM was achieved without any adverse effects in 457 of 500 corneas (91.4%). In 32 corneas (6.4%) with micro-tears during preparation, stripping from the opposite side was possible. However, 11 of the 500 corneas (2.2%) showed extremely strong adhesion leading to multiple tears of DM and preventing successful preparation of the graft. Endothelial cell number, donor age, post-mortem time, time in culture and pigmentation of the trabecular meshwork showed no significant correlation with failure to successfully obtain a DM graft. Complicated graft preparations of one eye showed a highly significant correlation with complicated graft preparations in the fellow eye. TEM analysis of failed grafts showed abnormal invasion of stromal parts into the DM, cell accumulation and pigmentation in the DM plane. CONCLUSION: Using curvilinear forceps for dissecting of the graft shows valid and reproducible results in the vast majority (97.8%) of donor corneas.


Subject(s)
Descemet Membrane/surgery , Descemet Stripping Endothelial Keratoplasty/methods , Endothelium, Corneal/transplantation , Fuchs' Endothelial Dystrophy/surgery , Tissue Donors , Tissue and Organ Harvesting/methods , Aged , Cell Count , Descemet Membrane/ultrastructure , Endothelium, Corneal/ultrastructure , Female , Fuchs' Endothelial Dystrophy/diagnosis , Graft Survival , Humans , Male , Microscopy, Electron, Transmission , Refraction, Ocular , Reproducibility of Results
9.
Hum Gene Ther Clin Dev ; 29(3): 121-131, 2018 09.
Article in English | MEDLINE | ID: mdl-30187779

ABSTRACT

Achromatopsia is an autosomal recessively inherited congenital defect characterized by a lack of cone photoreceptor function, leading to severely impaired vision. In this clinical study, achromatopsia patients were treated with a single subretinal injection of rAAV.hCNGA3 to restore cone function. The focus of this trial was on the safety of the treatment. After surgery, patients were monitored in eight extensive visits during the first year, followed by a 4-year follow-up period with annual visits. For essential complementation of the standard ophthalmological and systemic examinations, disease-specific methods were developed to assess the safety, efficacy, and patient-reported outcomes in this trial.


Subject(s)
Color Vision Defects/genetics , Color Vision Defects/therapy , Cyclic Nucleotide-Gated Cation Channels/genetics , Genetic Therapy/adverse effects , Adult , Aged , Color Vision Defects/pathology , Cyclic Nucleotide-Gated Cation Channels/administration & dosage , Cyclic Nucleotide-Gated Cation Channels/adverse effects , Dependovirus/genetics , Dose-Response Relationship, Drug , Female , Genetic Vectors/administration & dosage , Humans , Injections , Male , Middle Aged , Mutation , Retinal Cone Photoreceptor Cells/drug effects , Retinal Cone Photoreceptor Cells/pathology
10.
Cornea ; 37(10): 1334-1336, 2018 Oct.
Article in English | MEDLINE | ID: mdl-30063478

ABSTRACT

PURPOSE: To describe a new surgical technique for performing Descemet membrane endothelial keratoplasty (DMEK) using a temporary hydrophilic methacrylate sheet in the anterior chamber in cases of previous vitrectomy with complete removal of the vitreous body. METHODS: Medical records were reviewed for baseline and follow-up best spectacle-corrected visual acuity, endothelial cell count, and indication for DMEK using a temporary hydrophilic methacrylate sheet. Operative notes were analyzed in all cases to identify any specific intraoperative event. RESULTS: Seven eyes of 7 patients were included in this analysis. Mean age was 65.7 ± 18.2 years, and the mean follow-up duration was 11.5 months. All 7 patients had previously received pars plana vitrectomy with complete vitreous removal. All patients were pseudophakic. For 5 patients, it was their first DMEK surgery and 2 patients had DMEK surgery for a second time, due to a failed outcome in previous surgery. Best spectacle-corrected visual acuity improved from 1.5 ± 0.5 logMAR before surgery to 0.76 ± 0.37 logMAR after surgery during follow-up (P < 0.001). The mean endothelial cell count was 2442 ± 304 cells/mm and decreased to 1484 ± 279 cells/mm during follow-up (P < 0.001). No complications were observed in any of the cases at the unfolding stage using this method. All grafts remained clear at the last follow-up visit. CONCLUSIONS: This modified approach of DMEK surgery, using a temporary hydrophilic methacrylate sheet for flattening the anterior chamber and facilitating graft unfolding, could help overcome possible complications of DMEK in completely vitrectomized eyes.


Subject(s)
Corneal Diseases/surgery , Descemet Membrane/surgery , Descemet Stripping Endothelial Keratoplasty/methods , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Methacrylates/therapeutic use , Middle Aged , Visual Acuity , Vitrectomy
11.
PLoS One ; 13(6): e0198793, 2018.
Article in English | MEDLINE | ID: mdl-29939996

ABSTRACT

PURPOSE: This study aimed to investigate the trends in the surgical methods and leading indications for corneal transplantations carried out over the last 12 years. METHODS: The data from the corneal graft waiting list and from all keratoplasties carried out between 2004 and 2015 at the University Eye Hospital in Tübingen were retrospectively analyzed. RESULTS: A total of 1,185 keratoplasties were performed between 2004 and 2015 at this hospital. The most common surgical indications for corneal transplantation were Fuchs' endothelial corneal dystrophy (35.2%) and keratoconus (18.9%) with keratoconus being the leading cause during early years (from 2004 to 2009) and Fuch's dystrophy being the leading cause from 2010 to 2015. Overall, the total count of performed keratoplasties increased, from 385 corneal transplantations during the first 6-year period to 800 corneal transplantations during the second 6-year period (P = 0.008, using Mann-Whitney test). The Descemet's membrane endothelial keratoplasty has become the favored surgical method for endothelial disorders with the number of Descemet's membrane endothelial keratoplasties increasing significantly from 2008 to 2015. This increasing trend was statistically significant (P < 0.001 using multivariate adaptive regression splines (MARS). A decreasing trend was also noted for the rate of penetrating keratoplasty since 2008 (P < 0.001 using MARS). CONCLUSIONS: This research showed major changes in the preferred corneal transplantation techniques and leading indications for keratoplasty over the last 12 years. More importantly, it seems that the rapid development and implementation of endothelial keratoplasty, especially the Descemet's membrane endothelial keratoplasty, has had a profound effect on and begun a new era in corneal transplantation.


Subject(s)
Corneal Transplantation/trends , Descemet Stripping Endothelial Keratoplasty/trends , Fuchs' Endothelial Dystrophy/surgery , Hospitals, University/trends , Keratoconus/surgery , Corneal Transplantation/methods , Corneal Transplantation/statistics & numerical data , Descemet Stripping Endothelial Keratoplasty/statistics & numerical data , Germany , Hospitals, University/statistics & numerical data , Humans , Retrospective Studies
12.
PLoS One ; 13(5): e0198114, 2018.
Article in English | MEDLINE | ID: mdl-29813111

ABSTRACT

BACKGROUND: No evidence-based therapy exists for non-arteritic central retinal artery occlusion (NA-CRAO). Retinal ischemic tolerance is low; irreversible damage occurs within four hours of experimental NA-CRAO. In previous randomized trials evaluating intra-arterial or intravenous thrombolysis (IVT) in NA-CRAO, only one patient was treated this early. In December 2013, the Departments of Neurology & Stroke and Ophthalmology at University Hospital Tuebingen, Germany, decided to treat patients using IVT within 4.5 hours of NA-CRAO, the therapeutic window established for ischemic stroke. MATERIALS AND METHODS: Consecutive NA-CRAO patients with severe visual loss received IVT after exclusion of intracranial hemorrhage. Follow-up was conducted at day 5 (d5) and day 30 (d30). Visual outcomes were compared to the conservative standard treatment (CST) arm of the EAGLE-trial. RESULTS: Until August 2016, 20 patients received IVT within 4.5 hours after NA-CRAO with a median onset-to-treatment time of 210 minutes (IQR 120-240). Visual acuity improved from baseline mean logarithm of the minimum angle of resolution 2.46±0.33 (SD) (light perception) to 1.52±1.09 (Snellen equivalent: 6/200) at d5 (p = 0.002) and 1.60±1.08 (Snellen equivalent: 6/240) at d30. Compared to the EAGLE CST-arm, functional recovery to reading ability occurred more frequently after IVT: 6/20 (30%) versus 1/39 (3%) at d5 (p = 0.005) and at d30 5/20 (25%) versus 2/37 (5%) (p = 0.045). Two patients experienced serious adverse events (one angioedema and one bleeding from an abdominal aortic aneurysm) but recovered without sequelae. CONCLUSIONS: IVT within 4.5 hours after symptom onset may represent an effective treatment of NA-CRAO. Randomized trials are warranted to evaluate efficacy and safety of early IVT in acute NA-CRAO.


Subject(s)
Retinal Artery Occlusion/therapy , Thrombolytic Therapy , Veins , Acute Disease , Aged , Female , Humans , Male , Prospective Studies , Safety , Thrombolytic Therapy/adverse effects
13.
Invest Ophthalmol Vis Sci ; 59(5): 1910-1915, 2018 04 01.
Article in English | MEDLINE | ID: mdl-29677353

ABSTRACT

Purpose: To study longitudinal changes of anti-drug antibody (ADA) titers to recombinant adeno-associated virus serotype 8 (rAAV8) capsid epitopes in nonhuman primates (NHP) and patients. Methods: Three groups of six NHP each received subretinal injections (high dose: 1 × 1012 vector genomes [vg], low dose: 1 × 1011 vg, or vehicle only). Four additional animals received intravitreal injections of the high dose (1 × 1012 vg). Three patients received 1 × 1010 vg as subretinal injections. ELISA quantified ADA levels at baseline and 1, 2, 3, 7, 28, and 90 days after surgery in NHP and at baseline and 1, 3, and 6 months after surgery in patients. Results: Two out of 22 animals lacked ADA titers at baseline and developed low ADA titers toward the end of the study. Titers in the low-dose group stayed constant, while two of six animals from the high-dose group developed titers that rose beyond the range of the assay. All animals from the intravitreal control group showed a rise in ADA titer by day 7 that peaked at day 28. Preliminary data from the clinical trial (NCT02610582) show no humoral immune response in patients following subretinal delivery of 1 × 1010 vg. Conclusions: No significant induction of ADA occurred in NHP when mimicking the clinical scenario of subretinal delivery with a clinical-grade rAAV8 and concomitant immunosuppression. Likewise, clinical data showed no humoral immune response in patients. In contrast, intravitreal delivery was associated with a substantial humoral immune response. Subretinal delivery might be superior to an intravitreal application regarding immunologic aspects.


Subject(s)
Capsid Proteins/immunology , Color Vision Defects/therapy , Cyclic Nucleotide-Gated Cation Channels/immunology , Dependovirus/genetics , Genetic Therapy , Immunity, Humoral/physiology , Animals , Antibodies, Viral/blood , Color Vision Defects/immunology , Enzyme-Linked Immunosorbent Assay , Female , Genetic Vectors/administration & dosage , Humans , Intravitreal Injections , Macaca fascicularis , Male , Retina/virology , Vitreous Body/virology
14.
Biomaterials ; 157: 98-106, 2018 03.
Article in English | MEDLINE | ID: mdl-29258013

ABSTRACT

Nucleic acids represent very appealing building blocks for the construction of nano-scaled objects with great potential applications in the field of drug delivery where multifunctional nanoparticles (NPs) play a pivotal role. One opportunity for DNA nanotechnology lies in the treatment of ophthalmic diseases as the efficacy of eye drops is impaired by the short survival time of the drug on the eye surface. As a consequence, topical administration of ocular therapeutics requires high drug doses and frequent administration, still rarely providing high bioavailability. To overcome these shortcomings we introduce a novel and general carrier system that is based on DNA nanotechnology. Non-toxic, lipid-modified DNA strands (12mers with 4 lipid modified thymines at the 5' end) form uniform NPs (micelles), which adhere to the corneal surface for extended periods of time. In a single self-assembly step they can be equipped with different drugs by hybridization with an aptamer. The long survival times of DNA NPs can be translated into improved efficacy. Their functionality was demonstrated in several ex-vivo experiments and in an in-vivo animal model. Finally, the NPs were confirmed to be applicable even for human tissue.


Subject(s)
DNA/administration & dosage , Drug Delivery Systems , Eye Diseases/therapy , Nanoparticles/administration & dosage , Ophthalmic Solutions/administration & dosage , Administration, Ophthalmic , Animals , Cornea/metabolism , DNA/chemistry , Eye Diseases/genetics , Female , Humans , Mice , Mice, Inbred C57BL , Micelles , Nanoparticles/chemistry , Swine
15.
Cell Tissue Bank ; 19(3): 269-276, 2018 Sep.
Article in English | MEDLINE | ID: mdl-29079991

ABSTRACT

Human corneas usually are not available for research, as they are used for transplantation only. At the same time, scientific studies on cultured human endothelial cells can produce misleading results due to inevitable dedifferentiation. Therefore, an organ-culture model of porcine corneas-displaying endothelial cell death rates comparable to those of cultured human corneas-would be very desirable. Fresh pig eyes were prepared under sterile conditions to obtain corneoscleral buttons, corneal buttons and so called "split corneal buttons" (new preparation method) and cultivated for 15 days. Morphology of the endothelial cell layer was observed by light microscopy on day 1, 8 and 15. On day 15 staining with trypan blue and alizarin red S was performed. Photographs were evaluated in a randomized, blinded manner. Here, the morphology of the corneal endothelium and the number of endothelial cells per mm2 were analyzed. After 15 days of cultivation the endothelial cell layer was maintained only in corneal buttons and split corneal buttons. Alizarin red S stained areas and the existence of polymorphisms like rosette figures and reformation figures were significantly less frequent in split corneal buttons than in corneal buttons. Loss of endothelial cells was significantly greater in corneal buttons [575 ± 25/250 cells/mm2 (median ± 25%/75%-quantile); 14.8%] than in split corneal buttons [417 ± 138/179 cells/mm2 (median ± 25%/75%-quantile); 10.2%]. The new preparation method of split corneal buttons allows the cultivation of porcine corneas for 2 weeks with cell death rates comparable to those of the corresponding human tissue in cornea banks without the need to add de-swelling additives to the media. This is therefore a simple and highly reliable method model to be applied in intervention studies on corneal endothelial cells in their natural compound.


Subject(s)
Endothelium, Corneal/cytology , Organ Culture Techniques/methods , Animals , Anthraquinones/analysis , Cell Count , Cell Death , Endothelium, Corneal/ultrastructure , Staining and Labeling/methods , Swine , Trypan Blue/analysis
16.
Invest Ophthalmol Vis Sci ; 58(13): 5792-5801, 2017 11 01.
Article in English | MEDLINE | ID: mdl-29117317

ABSTRACT

Purpose: To investigate shedding and biodistribution characteristics of recombinant adeno-associated virus serotype 8 (rAAV8) after single-dose subretinal or intravitreal injection in nonhuman primates (NHP, Macaca fascicularis) as a surrogate for environmental hazard and patient safety. Methods: In a study for regulatory submission, 22 NHP were divided into four cohorts receiving either single subretinal injections of vehicle or clinical grade rAAV8 (1 × 1011 or 1 × 1012 vector genomes [vg]) versus single intravitreal application of 1 × 1012 vg. Viral shedding and biodistribution were monitored in biofluids for up to 91 days, followed by necropsy and tissue harvesting of all major organs, the visual pathway, and lymphatic tissue. Quantification of vector genomes was done by quantitative (q)PCR. Results: Shedding occurred in a dose-dependent manner in all biofluids and persisted for a maximum of 7 days. Intravitreal delivery led to increased and persistent (up to 13 weeks) distribution of vector genomes in blood and draining lymphatic tissue, increased off-target deposition, and inefficient gene transfer to the retina. No vector targeting of the germ line was observed in any cohort. Conclusions: These data illustrate that subretinal application of rAAV8 leads to a more favorable biodistribution profile compared to intravitreal injections. Extraocular biodistribution is limited after subretinal delivery, while intravitreal injection leads to both greater and more persistent systemic exposure, evident in blood and lymphatic tissues. With the knowledge on the dynamics of shedding in a setting mimicking clinical application, guidelines can be developed to refine clinical trial protocols to reduce the risk for trial subjects and their environment.


Subject(s)
Dependovirus/genetics , Genetic Therapy/methods , Genetic Vectors , Recombinant Proteins/administration & dosage , Retinal Diseases/therapy , Animals , Dose-Response Relationship, Drug , Female , Intravitreal Injections , Macaca fascicularis , Male , Retina , Retinal Diseases/metabolism , Tissue Distribution
17.
Front Neurosci ; 11: 445, 2017.
Article in English | MEDLINE | ID: mdl-28878616

ABSTRACT

Purpose: We assessed the safety and efficacy of a technically advanced subretinal electronic implant, RETINA IMPLANT Alpha AMS, in end stage retinal degeneration in an interim analysis of two ongoing prospective clinical trials. The purpose of this article is to describe the interim functional results (efficacy). Methods: The subretinal visual prosthesis RETINA IMPLANT Alpha AMS (Retina Implant AG, Reutlingen, Germany) was implanted in 15 blind patients with hereditary retinal degenerations at four study sites with a follow-up period of 12 months (www.clinicaltrials.gov NCT01024803 and NCT02720640). Functional outcome measures included (1) screen-based standardized 2- or 4-alternative forced-choice (AFC) tests of light perception, light localization, grating detection (basic grating acuity (BaGA) test), and Landolt C-rings; (2) gray level discrimination; (3) performance during activities of daily living (ADL-table tasks). Results: Implant-mediated light perception was observed in 13/15 patients. During the observation period implant mediated localization of visual targets was possible in 13/15 patients. Correct grating detection was achieved for spatial frequencies of 0.1 cpd (cycles per degree) in 4/15; 0.33 cpd in 3/15; 0.66 cpd in 2/15; 1.0 cpd in 2/15 and 3.3 cpd in 1/15 patients. In two patients visual acuity (VA) assessed with Landolt C- rings was 20/546 and 20/1111. Of 6 possible gray levels on average 4.6 ± 0.8 (mean ± SD, n = 10) were discerned. Improvements (power ON vs. OFF) of ADL table tasks were measured in 13/15 patients. Overall, results were stable during the observation period. Serious adverse events (SAEs) were reported in 4 patients: 2 movements of the implant, readjusted in a second surgery; 4 conjunctival erosion/dehiscence, successfully treated; 1 pain event around the coil, successfully treated; 1 partial reduction of silicone oil tamponade leading to distorted vision (silicon oil successfully refilled). The majority of adverse events (AEs) were transient and mostly of mild to moderate intensity. Conclusions: Psychophysical and subjective data show that RETINA IMPLANT Alpha AMS is reliable, well tolerated and can restore limited visual functions in blind patients with degenerations of the outer retina. Compared with the previous implant Alpha IMS, longevity of the new implant Alpha AMS has been considerably improved. Alpha AMS has meanwhile been certified as a commercially available medical device, reimbursed in Germany by the public health system.

18.
Acta Ophthalmol ; 95(8): e706-e712, 2017 Dec.
Article in English | MEDLINE | ID: mdl-28321999

ABSTRACT

PURPOSE: To assess the influence of donor, environment and storage factors on the contamination rate of organ-cultured corneas, to consider the microbiological species causing corneal contamination and to investigate the corresponding sensitivities. METHODS: Data from 1340 consecutive donor corneas were analysed retrospectively. Logistic regression analysis was used to assess the influence of different factors on the contamination rate of organ-cultured corneas for transplantation. RESULTS: The mean annual contamination rate was 1.8 ± 0.4% (range: 1.3-2.1%); 50% contaminations were of fungal origin with exclusively Candida species, and 50% contaminations were of bacterial origin with Staphylococcus species being predominant. The cause of donor death including infection and multiple organ dysfunction syndrome increased the risk of bacterial or fungal contamination during organ culture (p = 0.007 and p = 0.014, respectively). Differentiating between septic and aseptic donors showed an increased risk of contamination for septic donors (p = 0.0020). Mean monthly temperature including warmer months increased the risk of contamination significantly (p = 0.0031). Sex, donor age, death to enucleation, death to corneoscleral disc excision and storage time did not increase the risk of contamination significantly. CONCLUSION: The genesis of microbial contamination in organ-cultured donor corneas seems to be multifactorial. The main source of fungal or bacterial contamination could be resident species from the skin flora. The rate of microbial contamination in organ-cultured donor corneas seems to be dependent on the cause of donor death and mean monthly temperature.


Subject(s)
Bacteria/isolation & purification , Cornea/microbiology , Corneal Transplantation , Eye Banks/statistics & numerical data , Eye Infections, Bacterial/epidemiology , Organ Culture Techniques/methods , Organ Preservation/methods , Adolescent , Adult , Aged , Aged, 80 and over , Cornea/cytology , Culture Media , Eye Infections, Bacterial/microbiology , Female , Follow-Up Studies , Germany/epidemiology , Humans , Male , Middle Aged , Prevalence , Retrospective Studies , Tissue Donors , Young Adult
19.
Acta Ophthalmol ; 95(8): e727-e733, 2017 Dec.
Article in English | MEDLINE | ID: mdl-27914117

ABSTRACT

PURPOSE: To evaluate the treatment of selected patients with uveal melanoma with endoresection and adjuvant ruthenium brachytherapy. METHODS: Thirty-five patients with uveal melanoma not suitable for ruthenium plaque monotherapy were treated with endoresection and adjuvant ruthenium brachytherapy between January 2001 and October 2013. Recurrence-free survival, globe retention, course of visual acuity (VA), occurrence of therapy-related complications and metastasis-free and overall survival were analysed retrospectively. RESULTS: Eight patients (22.9%) had a tumour recurrence after a median follow-up of 49.5 months (range: 21-134 months). Enucleation was necessary in eight patients. Thirty-two patients (91%) had a loss of VA with a median loss of nine lines (range: 0 to -39 lines); VA was stable in three patients and no patients had a gain in VA. Four patients (11.4%) developed radiation retinopathy. Metastases were detected in seven patients (20.0%) during follow-up. The occurrence of metastasis was significantly associated with monosomy 3 (p < 0.0001). Twenty-four patients (68.6%) were alive at the end of follow-up. Five patients (14.3%) died because of uveal melanoma (UM) metastasis. CONCLUSIONS: Endoresection with adjuvant ruthenium brachytherapy is an option for selected patients with UM who cannot be treated with brachytherapy as monotherapy. About two-thirds of eyes can be retained long term without recurrences. Visual acuity cannot be maintained in most cases, and may even decrease considerably. Radiation complications are comparatively rare and not a significant problem.


Subject(s)
Brachytherapy/methods , Melanoma/therapy , Ruthenium Radioisotopes/therapeutic use , Uveal Neoplasms/therapy , Aged , Aged, 80 and over , Disease-Free Survival , Female , Follow-Up Studies , Germany/epidemiology , Humans , Male , Melanoma/diagnosis , Melanoma/mortality , Middle Aged , Ophthalmologic Surgical Procedures , Radiotherapy, Adjuvant , Retrospective Studies , Survival Rate/trends , Time Factors , Treatment Outcome , Uveal Neoplasms/diagnosis , Uveal Neoplasms/mortality , Visual Acuity
20.
Acta Ophthalmol ; 95(1): e1-e9, 2017 Feb.
Article in English | MEDLINE | ID: mdl-27576860

ABSTRACT

PURPOSE: Glaucoma filtration surgery (GFS) fails due to fibrosis. The α5ß1-integrin plays a pivotal role in fibrosis, angiogenesis and inflammation. This is the first experiment evaluating the prevention of fibrosis after GFS by a specific small molecule α5ß1-integrin inhibitor (CLT-28643). METHODS: Twenty-four rabbits received trabeculectomy on their right eyes. The rabbits were randomized into three groups of eight eyes each. CLT-28643 was given as a single subconjunctival injection intraoperatively to two of the right eye groups followed by postoperative vehicle eye drops (CLT+ group) or CLT-28643 eye drops 4 times daily (CLT++ group). A third group received mitomycin-C (MMC) intraoperatively (sponge application, 0.04%, 2 min) followed by vehicle eye drops postoperatively. The control-surgery group consisted of 12 left eyes having trabeculectomy with no adjunctive therapy. The remaining 12 left eyes formed the untreated group. Clinical assessment included intraocular pressure (IOP) measurement, slit-lamp examination (including bleb survival and morphology) and bleb photography. The rabbits were killed after four weeks for histology. RESULTS: Both CLT-28643-treated groups showed significantly prolonged bleb survival, and better bleb score compared to the control-surgery group. At end of the study, most functioning blebs were found in the MMC group (MMC group 75%; CLT+ group 12.5%, CLT++ group 25%; CLT+ group 12.5%, control-surgery group 0%). CLT-28643 was non-toxic and well tolerated. CONCLUSIONS: This rabbit GFS study indicates that inhibition of α5ß1-integrin by the novel α5ß1-integrin antagonist CLT-28643 significantly improved the outcome. The effect of a single intro-operative application of CLT-28643 seems to be inferior to 0.04% MMC.


Subject(s)
Aminoquinolines/pharmacology , Conjunctiva/pathology , Disease Models, Animal , Integrin alpha5beta1/antagonists & inhibitors , Surgical Stomas , Trabeculectomy , Wound Healing/drug effects , Administration, Topical , Alkylating Agents/administration & dosage , Animals , Collagen/metabolism , Conjunctiva/metabolism , Fibrosis/prevention & control , Glaucoma/surgery , Injections, Intraocular , Intraocular Pressure/drug effects , Male , Mitomycin/administration & dosage , Ophthalmic Solutions , Rabbits
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