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Purpose: To investigate the effect of Selective Retina Therapy (SRT) on inflammatory key factors such as complement factor-C3 (CC3), tumor growth factor-beta2 (TGF-β2), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ)., Materials and Methods: Porcine RPE-Bruch's membrane-choroid explants were irradiated with two SRT laser systems, SRT YLF and SRT YAG (Nd : YLF laser, wave length 527 nm, pulse duration 1.7 µs and Nd : YAG laser, wave length 532 nm, pulse duration 2.4 - 3 µs). Laser irradiation was performed on a spot size of 200 × 200 µm, 30 pulses, with a repetition rate of 100 Hz, and a radiant exposure of 140 (threshold RPE death) and 180 mJ/cm 2 per pulse (above threshold RPE death). Explants were cultivated in modified Ussing chambers and culture viability was assessed by calcein-AM cell staining. Secretion of inflammatory factors was analyzed by ELISA. Protein expression of tissue explants was assessed by Western blot., Results: Regeneration of RPE was observed after 4 days. One day after SRT with 140 mJ/cm 2 per pulse the secretion of basal CC3 decreased in ELISA. Following 180 mJ/cm 2 radiant exposure, the level of IFN-γ decreased at day 4., Conclusion: SRT does not induce the release of the pro-inflammatory factors analyzed in this in-vitro study., Competing Interests: Die Autoren geben an, dass kein Interessenkonflikt besteht., (Georg Thieme Verlag KG Stuttgart · New York.)

The purpose of this study was to characterise an ex-vivo adult porcine retina-retinal pigment epithelium (RPE) perfusion organ culture model. Fresh porcine full-thickness retina-RPE-choroid tissue samples were clamped into tissue carriers and mounted in two-compartment containers. The retinal and choroidal sides were continuously perfused with culture medium. pO(2), [Na(+)], [K(+)], [Cl(-)], [glucose], [lactate], and pH were measured in the medium. Tissue samples were examined after 24h, 4, 7, and 10 days in culture. The morphology of the retina and the RPE was examined by light and electron microscopy (LM, EM). The retinal cellular integrity was further examined by immunohistochemistry (Ki 67, GFAP, rhodopsin, synaptophysin, syntaxin, NF 200, TUNEL-test). Fresh porcine full-thickness retina-RPE-choroid tissue samples and tissue samples in static organ culture served as controls. LM, EM, and immunohistochemistry showed intact retinal and RPE cytoarchitecture kept in perfusion culture. Photoreceptor outer segments showed first signs of degeneration after 24h, significant signs of apoptosis and necrosis appeared in the retina after 4 days in perfusion culture. Control tissue samples kept in static culture showed disintegration of the retinal cytoarchitecture after 4 days in culture. The data show that adult porcine retina-RPE tissue can be maintained morphologically intact in perfusion organ culture for at least 10 days. Although first signs of degeneration set in after 24h the structural preservation of the tissue in perfusion organ culture is superior to that in static culture. The perfusion culture model of the retina refines organotypic in vitro test systems and may help to reduce the number of necessary animal experiments in retina and RPE research. It offers new perspectives for the safety testing of substances designed for intraocular application.

Purpose: To assess retinal toxicity of indocyanine green (ICG) in a porcine ex vivo perfusion organ culture model, and to measure intraretinal penetration of ICG applied to the retinal surface., Methods: The retinal surface of fresh porcine retinal tissue was exposed to ICG 0.1% and 1% dissolved in glucose 5% for 1 and 30 minutes with and without concomitant illumination. Specimens were then kept in perfusion organ culture for 24 hours before examination by light microscopy and the TUNEL technique. Tissue samples treated with DNAse served as positive controls, and samples exposed to saline served as negative controls. Fluorescence microscopy was used to localize ICG at 1 minute, 60 minutes, 2 hours, and 3 hours after a 1-minute exposure of the retinal surface to ICG 1%., Results: No increase in TUNEL-positive cells was observed after exposure to ICG 0.1% for 1 minute. Moderate apoptosis was found after 1-minute exposure to ICG 1% and 30-minute exposure to ICG 0.1%, and severe apoptosis was found after 30-minute exposure to ICG 1%. Concomitant application of light did not influence the degree of apoptosis. No signs of cell necrosis were found. After 1-minute exposure of the retinal surface, ICG 1% gradually penetrated the entire retina., Conclusions: ICG induced apoptosis but not necrosis in all nuclear retinal layers in a dose-dependent manner. Brief exposure to ICG 0.1% for 1 minute and illumination for 3 minutes simulated the intraoperative use of ICG. No retinal apoptosis or necrosis was observed. ICG briefly applied to the retinal surface gradually penetrated the entire retina.

There is uncertainty surrounding the safety of the vital stains currently used to assist macular surgery, and there may be other agents that are more suitable. This study aimed to validate a method of screening retinal vital stains for their potential surgical utility. Bovine retina was exposed to test agents at a range of concentrations. Masked observers determined the minimum dye concentration that reliably stained the retina, defined as the minimum visible concentration (MVC). Computer image analysis (CIE94 colour difference equation) was used to estimate the magnitude of the colour difference between stained and unstained retina. Agents that had favourable staining characteristics underwent safety testing using a retinal pigment epithelium and glial cell culture model. Cells were exposed to each agent and viability was assessed with a mitochondrial enzyme (MTT) assay, and fluorescent live-dead probe (ethidium homodimer-1/calcein-AM). Frozen sections were used to determine which retinal layers were stained. Techniques were tested on the following agents: alcian blue; diethyloxadicarbocyanine; Evan's blue; fast green; fluorescein; Janus green; methylene blue; naphthol green; neutral red; procian (reactive) yellow; rose bengal; and trypan blue. For most dyes, the results of image analysis showed that colour differences increased linearly with dye concentration, although some displayed a more exponential relationship. Five agents showed favourable staining characteristics: Evan's blue, rose bengal, naphthol green, neutral red, and trypan blue (MVC 0.02, 0.01, 0.1, 0.002, 0.01%, respectively). Safety testing of these five agents did not show toxicity, except in glial cells exposed to rose bengal. Relative to the negative control (saline), these showed a 48% reduction in viability using the MTT assay (p<0.001; t=4.71; CI 30-75%), and qualitative damage on fluorescence microscopy. Frozen sections showed that some agents produced diffuse staining of all retinal layers, others produced selective inner retinal staining. There are thousands of biological stains available and many of these may be more effective or safer than those currently used for retinal surgery. This study provides a means of screening potentially useful vital stains.

Purpose: To characterize the Michaelis-Menten kinetics of the taurine transporter (TT) in retinal pigment epithelium (RPE) freshly isolated from human donor eyes. To identify the rate limiting compartment in the pathway of taurine delivery from the choroidal blood supply to the outer retina composed by Bruch's-choroid (BC) and the RPE in the human older age group., Methods: In human donor samples (4 melanoma-affected eyes, and 14 control eyes; age range, 62-93 years), radiochemical techniques were used to determine the RPE taurine accumulation at various exogenous concentrations. The transport capability of human RPE was obtained from a kinetic analysis of the high-affinity carrier over a substrate concentration of 1 to 60 microM taurine., Results: Uptake of taurine into human RPE at a taurine concentration of 1 microM was independent of donor age (P > 0.05) and averaged at 2.83 +/- 0.27 (SEM) pmol/10 minutes per 6-mm trephine. Taurine transport by human RPE was mediated by a high-affinity carrier of K(m) 50 microM and V(max) of 267 pmol/10 minutes per 5-mm disc., Conclusions: In human donor RPE, uptake of taurine remained viable in the age range 62 to 93 years. Taurine transport rates in the RPE were lower than across the isolated BC complex, and thus the data suggest that the former compartment houses the rate-limiting step in the delivery of taurine to the outer retina.

Purpose: To assess the relative resistance presented individually by Bruch's membrane-choroid (BC) and the retinal pigment epithelium (RPE) to movement of taurine between the choroidal circulation and the outer retina. To quantify the effect of light-evoked changes in subretinal potassium concentration on the transepithelial transport of taurine across bovine RPE., Methods: Transport studies were performed in Ussing chambers with intact and RPE-denuded specimens of BC. RPE viability was monitored by recording transepithelial potential (TEP) and transepithelial resistance (TER). Taurine transport with substrate concentrations in the micro- and millimolar range, reflecting physiological taurine concentrations in plasma, retina, and subretinal space was quantified by high-performance liquid chromatography (HPLC) and radiotracer techniques. Taurine transport was also assessed after apical potassium concentration was lowered from 6.0 to 2.2 mM to mimic the effects of light., Results: Transport of taurine across RPE-BC at a 10-mM substrate concentration increased from 32.92 before to 111.72 nanomoles/4 mm per hour after removal of the RPE. Similarly, at 50 microM taurine, transport rates increased from 0.158 to 0.439 nanomoles/4 mm per hour after removal of the RPE. At both high (10 mM) and low (50 microM) substrate concentrations, lowering of apical potassium was associated with decreased transport of taurine across the RPE. For taurine concentrations greater than 42 microM, the rate-limiting compartment for transport of taurine to the outer retina was the RPE monolayer. Similar rates were observed across each compartment for concentrations <42 microM., Conclusions: The magnitude and directionality of taurine transport across the RPE is determined solely by the driving taurine concentration gradient and is modulated by subretinal levels of potassium. Such modulation may provide a mechanism for conserving retinal taurine. Processes that increase the resistance to diffusion across Bruch's membrane such as human ageing and increased thickening and deposition of debris associated with age-related macular degeneration (AMD) are likely to affect transport across the RPE, culminating in a secondary retinal taurine deficiency.

Purpose: To determine the relative influence of path length and matrix components on the movement of small solutes and water between the choroid and the outer retina., Methods: Human and bovine Bruch's membrane-choroid (BC) tissue samples were mounted in modified Ussing chambers, and the diffusion of taurine and hydraulic conductivity (Lp) was determined. In humans, diffusion of taurine was determined as a function of age of the donor. The relative contribution of Bruch's membrane in the BC complex to transport processes was measured after its removal by laser ablation. Similarly, the effect of choroidal path length was determined. In humans, tracking the trend of age-related thinning provided samples of various path lengths. In young bovine animals (< or =2 years old), choroidal thickness was adjusted by laser ablation., Results: Diffusion of taurine across human BC decreased linearly from 162.7 to 105.9 nanomoles/h per 3 mm between 10 and 90 years of age (P < 0.05). Ablation of Bruch's membrane increased diffusion of taurine from 129 to 287.9 nanomoles/h per 4 mm in human (donor age 55, 74, and 82 years; P < 0.005) but caused no statistically significant change in bovine BC. Diffusion of taurine across bovine BC was greater in samples with partially ablated choroid (218 nanomoles/h per 4 mm) than in normal control samples (128.75 nanomoles/h per 4 mm). Lp was not measurable in bovine samples after complete ablation of Bruch's membrane, but did not change significantly as the choroid thinned., Conclusions: The data suggest that both path length and matrix components contribute to the decline of diffusion of small solutes across BC with age. The importance of matrix components was also demonstrated in restricting the movement of water while choroidal thickness played little if any role.

Purpose: To modify existing experimental models to simulate the typical clinical presentation of human rhegmatogenous retinal detachment (RRD), namely an RD caused by a retinal break, in a phakic eye, with a posterior vitreous detachment (PVD); to model RRD in a species that is anatomically similar to humans; and to characterize the glial cell response to RRD., Methods: Mixed-breed pigs underwent vitrectomy, PVD, subretinal injection of viscoelastic, and creation of a break at the apex of the RD. The crystalline lens was not removed. Follow-up was for 0, 1, and 7 days. Tissue was processed for light and electron microscopy. The glial cell response was characterized using antibodies to glial fibrillary acidic protein (GFAP)., Results: Of 11 RRDs created in seven pigs, 10 increased in size and 1 decreased. Light and electron microscopy demonstrated typical features of RD. There was constitutive expression of GFAP in astrocytes and Müller cells with increased immunoreactivity from day 1., Conclusions: This study provided a model of RRD that simulates the typical clinical presentation in humans. It used techniques that most vitreoretinal surgeons are familiar with, and an animal that is widely available and anatomically similar to humans. Anatomic success was high, and the glial cell response established comparability with other species.

Purpose: To determine the maximum size of molecule capable of freely diffusing across human retina, referred to as the retinal exclusion limit (REL), and the location of any sites of high resistance to diffusion. To assess the degree of interspecies variation in the REL of three animals commonly used to model human disease., Methods: Trephines of human neuroretina were mounted in a modified Ussing chamber. FITC-dextrans of various molecular weights (MWT) were dissolved in phosphate-buffered saline, and the rate of transretinal diffusion was determined over 24 hours with a spectrophotometer. The theoretical REL was calculated by extrapolating the linear relationship between the rate of diffusion and log(MWT). In separate experiments to determine the sites of barrier to diffusion, FITC-dextrans with a MWT greater than the calculated REL were applied to either the inner or outer retinal surface, processed as frozen sections, and viewed with a fluorescence microscope. Experiments to determine the REL were repeated in bovine, porcine, and rabbit retina., Results: The REL in human tissue was 76.5+/-1.5 kDa (6.11+/-0.04 nm). The inner and outer plexiform layers formed the sites of highest resistance to diffusion. The REL in pigs, cattle, and rabbits were 60 +/- 11.5, 78.5 +/- 20.5, and 86 +/- 30 kDa, respectively (5.68 +/- 0.45, 6.18 +/- 0.61, and 6.38 +/- 0.88 nm)., Conclusions: In humans, the inner and outer plexiform layers are sites of high resistance to the diffusion of large molecules, resulting in an REL of 76.5 kDa. There was only moderate interspecies variation in the REL of the animals studied, suggesting that they provide adequate models for the study of human transretinal macromolecular diffusion.

Purpose: This study was conducted to evaluate the effect of pupillary dilation on retinal nerve fiber layer (RNFL) thickness as measured by scanning laser polarimetry (SLP) in cataractous and noncataractous eyes., Methods: The study included 31 eyes of 31 consecutive patients (mean age, 62.5 +/- 14.0 years; range, 30-76 years). Eyes with refractive error exceeding 5.0 D sphere or 2.0 D cylinder, nonlenticular media opacity, cup-to-disc ratio > 0.9, corneal disease, ocular inflammation, or previous intraocular surgery were excluded. A standard reticule was used to measure pupillary diameter. Cataract grade was evaluated by a single observer using the Lens Opacities Classification System (LOCS III). RNFL thickness measurements were obtained by means of SLP before and after pupillary dilation., Results: Of the patients, 10 had clear lenses and 21 had variable degrees of lenticular opacification. In four eyes, imaging could not be performed because of dense cataracts. Mean pupillary diameters before and after dilation were 2.5 +/- 0.7 mm and 7.3 +/- 1.1 mm, respectively. There were no significant differences in global RNFL thickness before and after dilation in noncataractous and cataractous eyes. Among cataractous eyes in which imaging was possible, there was no correlation between difference in RNFL thickness before and after dilation and nuclear opalescence, nuclear color, and cortical and posterior subcapsular grading of the LOCS III score. Six of 27 eyes (22.2%) had a change of more than 10% in RNFL thickness after pupillary dilation., Conclusion: Although pharmacologic mydriasis does not statistically alter RNFL thickness as measured by SLP, approximately one fifth of such eyes will have a change of more than 10% in retardation. Uniformity in pupil size is recommended when longitudinally evaluating RNFL measurements.