Karl U. Bartz-Schmidt

Subretinal electronic chips allow blind patients to read letters and combine them to words

A light-sensitive, externally powered microchip was surgically implanted subretinally near the macular region of volunteers blind from hereditary retinal dystrophy. The implant contains an array of 1500 active microphotodiodes (‘chip’), each with its own amplifier and local stimulation electrode. At the implants tip, another array of 16 wire-connected electrodes allows light-independent direct stimulation and testing of the neuron–electrode interface. Visual scenes are projected naturally through the eyes lens onto the chip under the transparent retina. The chip generates a corresponding pattern of 38 × 40 pixels, each releasing light-intensity-dependent electric stimulation pulses. Subsequently, three previously blind persons could locate bright objects on a dark table, two of whom could discern grating patterns. One of these patients was able to correctly describe and name objects like a fork or knife on a table, geometric patterns, different kinds of fruit and discern shades of grey with only 15 per cent contrast. Without a training period, the regained visual functions enabled him to localize and approach persons in a room freely and to read large letters as complete words after several years of blindness. These results demonstrate for the first time that subretinal micro-electrode arrays with 1500 photodiodes can create detailed meaningful visual perception in previously blind individuals.

Safety, penetration and efficacy of topically applied bevacizumab: evaluation of eyedrops in corneal neovascularization after chemical burn

Purpose:  That vascular endothelial growth factor (VEGF) plays a major role in inflammatory angiogenesis has been well established. This pilot study was designed to evaluate experimental treatment with bevacizumab eyedrops in corneal neovascularization induced by alkali burn. The feasibility of topical administration, corneal cell viability and corneal penetration were investigated in an animal model.

SAFETY PROFILE OF BEVACIZUMAB ON CULTURED HUMAN CORNEAL CELLS

Purpose: To study the corneal biocompatibility of bevacizumab on various cultured human corneal cells. Methods: Cell cultures of corneal keratinocytes (CKs), corneal fibroblasts (CFs), and corneal endothelial cells (CECs) were harvested from human donor eyes and exposed to various concentrations of bevacizumab (0.25-5.0 mg/mL). Cell viability was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at days 1 and 4 after exposure. For cytotoxicity testing, confluent cells were cultured in serum-depleted medium, and the MTT test was performed after 24 hours of incubation. Expression of vascular endothelial growth factor (VEGF), VEGF receptors (VEGFR1 and VEGFR2), keratan sulphate (KS), and cytokeratin-3 (AE5) was studied by immunohistochemistry. Live/dead viability/cytotoxicity assay was performed and analyzed by fluorescence microscopy after 24 hours of incubation. Cell morphology was assessed with a phase-contrast microscope after 7 days of exposure with different concentrations of bevacizumab (0.25-5.0 mg/mL), and signs of cellular damage were assessed. Results: No cytotoxic effect of bevacizumab on CKs, CFs, and CECs could be observed when used at a concentration of 5.0 mg/mL or lower. Bevacizumab-treated cells showed no signs of cellular damage compared with the control. CKs, CFs, and CECs stained positively for VEGF, VEGFR1, and VEGFR2. CKs and CECs stained positively for AE5, whereas CFs were immunopositive for KS. Conclusions: Bevacizumab is not toxic to corneal cells of human origin in vitro at doses usually used for treatment of corneal neovascularization, which is 20-fold higher than that used for intravitreal application.

Antiproliferative and cytotoxic properties of bevacizumab on different ocular cells

Aim: To evaluate the antiproliferative and cytotoxic properties of bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), on human retinal pigment epithelium (ARPE19) cells, rat retinal ganglion cells (RGC5), and pig choroidal endothelial cells (CEC). Methods: Monolayer cultures of ARPE19, RGC5, and CEC were used. Bevacizumab (0.008–2.5 mg/ml), diluted in culture medium, was added to cells that were growing on cell culture dishes. Cellular proliferative activity was monitored by 5′-bromo-2′-deoxyuridine (BrdU) incorporation into cellular DNA and the morphology assessed microscopically. For cytotoxicity assays ARPE19, RGC5, and CEC cells were grown to confluence and then cultured in a serum depleted medium to ensure a static milieu. The MTT test was performed after 1 day. The “Live/Dead” viability/cytotoxicity assay was performed and analysed by fluorescence microscopy after 6, 12, 18, 24, 30, 36, and 48 hours of incubation. Expression of VEGF, VEGF receptors (VEGFR1 and VEGFR2) and von Willebrand factor was analysed by immunohistochemistry. Results: No cytotoxicity of bevacizumab on RGC5, CEC, and ARPE19 cells could be observed after 1 day. However, after 2 days at a bevacizumab concentration of 2.5 mg/ml a moderate decrease in ARPE19 cell numbers and cell viability was observed. Bevacizumab caused a dose dependent suppression of DNA synthesis in CEC as a result of a moderate antiproliferative activity (maximum reduction 36.8%). No relevant antiproliferative effect of bevacizumab on RGC5 and ARPE19 cells could be observed when used at a concentration of 0.8 mg/ml or lower. CEC and ARPE 19 cells stained positively for VEGF, VEGFR1, and VEGFR2. More than 95% of the CEC were positive for von Willebrand factor. Conclusions: These experimental findings support the safety of intravitreal bevacizumab when used at the currently applied concentration of about 0.25 mg/ml. Bevacizumab exerts a moderate growth inhibition on CEC when used in concentrations of at least 0.025 mg/ml. However, at higher doses (2.5 mg/ml) bevacizumab may be harmful to the retinal pigment epithelium.

Efficacy of rho-kinase inhibition in promoting cell survival and reducing reactive gliosis in the rodent retina.

PURPOSE To analyze the outcomes of Rho-kinase (ROCK) inhibition on retinal cell survival and glial reactivity under adverse conditions. METHODS Organotypic cultures of mouse retinas were incubated with the specific ROCK-inhibitor H-1152P for 24 to 48 hours under serum deprivation. Cell damage was determined by ethidium homodimer-1 uptake and caspase-3 cleavage. Immunohistochemistry and Western blot were performed to detect reactive gliosis and to confirm the specificity of H-1152P. The cytokine profile of the culture medium was analyzed using a membrane-based array. H-1152P was administered intravitreally into rats before optic nerve crush (ONC) and the extent of apoptosis and reactive gliosis was determined after 7 days. RESULTS Cell damage in cultured retinas was significantly reduced in response to 1 microM H-1152P, particularly in the ganglion cell layer. This was associated with a decrease in the levels of glial fibrillary acidic protein (GFAP) isoforms and the number of reactive astrocytes, Müller cells, and microglia. The release of proinflammatory cytokines including TNF-alpha, interferon-gamma, and IL-6 was also reduced, which likely contributed to the significantly lower toxicity of the conditioned media collected from retinas incubated with H-1152P. H-1152P (1 microM) suppressed the ROCK-dependent phosphorylation of adducin without a considerable interference with the protein kinase A/C-mediated phosphorylation events, indicating the specificity of the inhibitor for ROCK. H-1152P also resulted in a significant decrease in the extent of apoptosis and reactive gliosis after ONC. CONCLUSIONS These results demonstrate the neuroprotective effect of H-1152P-mediated ROCK-inhibition on retinal cells under stress, which may rely partly on the attenuation of glial cell reactivity.

Quantitative Morphologic and Functional Evaluation of the Optic Nerve Head in Chronic Open-Angle Glaucoma

Glaucoma leads to morphologic changes of the optic nerve head and to functional defects. Morphologic changes in the three-dimensional surface structure of the optic nerve head at its entrance site into the globe can be examined by laser scanning tomography. The standard technique for evaluating functional defects in glaucoma is static computerized perimetry. We compared these two techniques to determine which is more sensitive for follow-up of glaucomatous damage of the optic nerve head. If decreased function is presumed to precede imminent cell death, visual field analysis should be the more sensitive method, as cell death results in absolute defects of the visual field. However, the neuronal networks do not necessarily function in this way. In the case of loss of individual elements in the neuronal network, the complex linkages, even at the retinal level, are able to maintain functions and compensate for loss of function, which means that visual field defects would not be prominent. If the damage increases with time and is accompanied by a progressive loss of ganglion cells, however, compensation is eventually no longer possible, and the functional defects then become measurable by visual field analysis. Thus, morphologic absolute changes may be more prominent than visual field defects in the early stages of glaucoma. To evaluate the quantitative relationship between morphometrically measurable defects of the optic nerve head and measurable functional defects, we first examined the visual field with static computerized perimetry and then evaluated the surface structure with a laser scanning tomograph in 90 patients with chronic open-angle glaucoma, 10 patients with ocular hypertension, and 10 patients without any eye disease. Based on the 95th percentile of the standardized rim/disk area ratio, we calculated the relative rim area loss and correlated this with the mean defect in visual field analysis. The scatterplot shows an exponential curve. In the early stages of glaucoma, visual field defects were less prominent than morphologic absolute changes; 40% of the neuroretinal rim area is lost by glaucomatous optic nerve damage before first defects in visual field analysis appear. In late stages of glaucoma, changes in perimetry are more prominent than those observed with biomorphometry. These results show that in the follow-up of patients with early stages of glaucomatous damage, special attention should be given to morphologic absolute changes. In patients with advanced glaucoma, progress of the damage should be observed by repeated functional, rather than morphologic, examinations. It is important to keep in mind, however, that the sensitivity of any method is dependent on technology. One reason why functional tests may not be as sensitive as morphologic examination in observing patients with early stages of glaucoma may simply be that functional tests are not yet sensitive enough to detect early damage.

Retinal pigment epithelial tears after single administration of intravitreal bevacizumab for neovascular age-related macular degeneration

PurposeTo analyse retinal pigment epithelial (RPE) tears following single administration of intravitreal bevacizumab for neovascular age-related macular degeneration (AMD) during early follow-up.MethodsInterventional, retrospective, non-comparative case series included 397 patients (409 eyes) of the 746 consecutive patients that met the eligibility criteria. Standardized visual acuity testing, fluorescein angiography, and optical coherence tomography were performed. Data collected included status of the fellow eye, previous treatment, subtypes of choroidal neovascularization (CNV), size and composition of the lesion. Multiple linear regression modelling was used to explore the effect of baseline parameters on the RPE tears. Primary end point was occurrence of RPE tears within 6 weeks after therapy.ResultsFifteen of the 409 eyes (3.6%) developed RPE tear (95% confidence interval: 2.2–6.0, odds ratio: 26.3). The statistical modelling showed significant association between RPE tear and occult without classic CNV/predominantly haemorrhage vspredominantly/minimal classic CNV (P=0.019), as well as medium or large (>4 disc area) vssmall size of the total lesion (P=0.038). Previous treatment and status of the fellow eye did not statistically influence the risk of RPE tears.ConclusionsAn RPE tear can develop in up to 3.6% of eyes with neovascular AMD following single administration of intravitreal bevacizumab in a short-term follow-up. Medium and large lesion size and occult without classic and predominantly haemorrhagic subtype of CNV were important predictive factors. Preoperative assessment of the lesion characteristics may help in identifying the risk of individual patients with neovascular AMD before intravitreal bevacizumab treatment.

Sutureless amniotic membrane fixation using fibrin glue for ocular surface reconstruction in a rabbit model.

Purpose: Amniotic membrane transplantation has become an important treatment option for corneal surface reconstruction. However, suture fixation of the transplant has various disadvantages like corneal irritation, scarring, graft loss due to membrane shrinkage, and the need for subsequent suture removal. Replacement of sutures by bioadhesives might be an advantageous alternative. This controlled study was designed to evaluate a new sutureless technique for amniotic membrane fixation onto the corneal surface by using fibrin glue. Methods: Standardized disks of cryopreserved amniotic membranes were transplanted onto the deepithelialized cornea of 12 rabbits using either conventional suture fixation or a new fibrin glue technique. The rabbits were followed-up with slit-lamp examination and fluorescein staining until epithelialization was completed. Consecutively, the rabbits were killed and the eyes processed for histology and immunohistochemistry for cytokeratin-3. Results: All membranes of both groups stayed in place throughout the follow-up time and showed a progressive graft epithelialization that was completed after 12 days. Whereas suture-fixated membranes showed progressive tissue shrinkage, fibrin-glued sheets remained unaltered. In the bioadhesive group, histology revealed a smooth fibrin layer in the graft-host interface and a continuous, stratified layer of cytokeratin-3 expressing corneal epithelial cells on the membrane surface. In contrast, suture-fixated membranes showed contracted and prominent membrane edges with epithelial ingrowth into the submembrane interface. Conclusion: Our results demonstrate the general feasibility of reproducible and reliable sutureless amniotic membrane fixation onto the corneal surface in rabbits. Stable adherence is maintained until epithelialization is completed. The sutureless technique gives sufficient manipulation time for the sheet before the final cross-linking process is completed. Furthermore, several advantageous characteristics could be demonstrated as increased biocompatibility, better epithelialization pattern and the lack of membrane shrinkage.

Combined treatment of a juxtapapillary retinal capillary haemangioma with intravitreal bevacizumab and photodynamic therapy

Combined treatment of a juxtapapillary retinal capillary haemangioma with intravitreal bevacizumab and photodynamic therapy

Clinicopathological findings of choroidal neovascularisation following verteporfin photodynamic therapy

Aims: To report the clinicopathologic findings of surgically excised choroidal neovascularisation (CNV) three days after verteporfin photodynamic therapy (PDT). Methods: In three patients (three eyes) with age related macular degeneration, the CNV was surgically removed three days after PDT. The CNV specimens were examined by light microscopy. Results: The patients had subfoveal classic CNV. Fluorescein angiography revealed non-perfusion of the CNV after PDT and before surgery in all eyes. The light microscopy of the CNV membranes showed swollen and damaged endothelium. Thrombus formation or vascular occlusion in the CNV vessels was not detected. Conclusion: PDT did not cause a thrombosis of the vessels within the CNV three days after PDT. Severe endothelial damage of the CNV was observed and is likely a primary effect of PDT. Non-perfusion of the CNV at this stage is possibly secondary to occlusion at a deeper level, namely the underlying feeding choroid.