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The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses’ vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies.

Adhesion and spreading of retinal pigment epithelial (RPE) cells on fibronectin-rich extracellular matrices is a crucial event in the pathogenesis of proliferative vitreoretinopathy (PVR). In the present study we explored the capacity of galectin-3, a β-galactoside-binding endogenous lectin, to inhibit early PVR-associated cellular events from a therapeutic perspective. We assessed the relative expression levels of galectin-3 in native RPE and dedifferentiated, cultured RPE. Galectin-3 was constitutively expressed under in vivo and in vitro conditions and was abundant in cultured cells. Treatment of human RPE cells with soluble galectin-3 disclosed no toxicity within control limits up to 250 μg/ml. When added to the medium, galectin-3 dose-dependently inhibited attachment and spreading of the cells on fibronectin by more than 75%. When coated on the plastic surface, galectin-3 alone impaired attachment and spreading of RPE cells, and reduced attachment but not spreading on fibronectin. Galectin-3 bound to the cell surface, and, as determined by the use of the competing sugar β-lactose, galectin-3-mediated effects were dependent on carbohydrate binding. To ascertain the role of the ability of galectin-3 to form pentamers, we proteolytically removed the N-terminal, cross-linking section. The remaining C-terminal carbohydrate-binding domain alone failed to bind to cells and was functionally inactive. These results emphasize the relevance of both properties, i.e., glycan-binding and cross-linking of glycan moieties, for the inhibitory activity of galectin-3. Incubation of mobilized RPE cells with galectin-3 significantly disturbed microfilament assembly and, in correlation with decreased attachment, inhibited ERK phosphorylation. Therefore, galectin-3, acting as a cross-linking lectin on the cell surface, negatively regulates attachment and spreading of RPE cells in vitro. This effect, at least in part, is attributed to an inhibition of the ERK-MAPK pathway, which prevents cytoskeletal rearrangements needed for RPE cell attachment and spreading. Further investigation at this pathway may disclose a promising nouveau perspective for treatment and prophylaxis of early PVR.

Purpose: Indocyanine green (ICG) is a commonly used vital dye for macular surgery. Recent reports implicate that its use might be associated with less favourable results regarding postoperative visual outcome and damage of retinal cells, and atrophic degeneration of the retinal pigment epithelium (RPE) has been described. This study investigates the effects of ICG on light-induced senescence of RPE cells. Methods: Primary human RPE cells were either pre-incubated with ICG in concentrations of 0.005% and 0.05% or not and then exposed to white light. After 10 min of irradiation viability, induction of intracellular reactive oxygen species (ROS) and senescence-associated β-galactosidase activity (SA β-Gal) were determined. Expression and secretion of matrix metalloproteinases (MMPs) 1 and 3 and their mRNA were determined by RT-PCR and ELISA. Results: Light exposure decreased RPE cell viability by 46%. Treatment with 0.005% and 0.05% ICG alone decreased RPE cell viability by 7% and 21%. In addition, expression of ROS, SA β-Gal, and MMP-1 and 3 was significantly increased. When 0.005% and 0.05% ICG treatments were combined with light exposure, viability decreased by 69% and 82% compared to the untreated control. Effects on the expression of ROS, SA β-Gal, and MMP-1 and 3 were, depending on the ICG dose, significantly increased when cells were pre-incubated with ICG and then illuminated. Conclusion: In this study, pretreatment with ICG significantly increased light-induced oxidative stress and senescence. This might indicate a potential, supplementary mechanism that could explain RPE alterations and reduced functional results after ICG-assisted internal limiting membrane peeling.

Purpose To compare the possible protective effects of the ultraviolet (UV)-filtering and blue light–filtering SN60AT intraocular lens (IOL) and the untinted UV-filtering SA60AT IOL with regard to light-induced stress on human retinal pigment epithelium (RPE). Setting Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. Methods Primary human RPE cells were exposed to white light, and a tinted or untinted IOL was placed in the light beam. After 15 to 60 minutes of irradiation, cell viability was determined by a colorimetric test (tetrazolium dye-reduction assay) and a microscopic live/dead assay. The expression of vascular endothelial growth factor-α (VEGF-α), Bax, and Bcl-2 and their mRNA was determined by reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting. Results Without an IOL, white-light exposure decreased cell viability compared with the decrease with the nonirradiated control in a time-dependent manner. Light-induced cell death was significantly reduced by both the tinted IOL and untinted IOL. The combined UV and blue-light filtering attenuated light-induced cell damage significantly more than UV filtering alone. Results of RT-PCR and Western blotting showed a significant time-dependent decrease in Bcl-2 and increase in Bax and VEGF-α that were significantly less with the tinted IOL than with the untinted IOL. Conclusions Both IOLs reduced light-induced RPE damage. The UV- and blue light–filtering IOL reduced damage more than the conventional IOL. This supports the hypothesis that blue light–filtering IOLs may prevent retinal damage in clinical use.

To evaluate the effect of intravitreal bevacizumab (Avastin; Genentech, Inc., South San Francisco, CA) injections on visual acuity and foveal retinal thickness in patients with central retinal vein occlusion (CRVO).In this prospective, noncomparative, consecutive, interventional case series, 46 patients received repeated intravitreal injections (1.25 mg) of bevacizumab. Main outcome measures were visual acuity (Snellen and ETDRS charts) and optical coherence tomography measurements in a 6-month follow-up period.Mean visual acuity improved from 20/250 at baseline to 20/80 at the 6-month follow-up (P0.001). ETDRS chart findings revealed a mean letter gain +/-SD from baseline to 6 months of 13.9 +/- 14.4 letters. Mean central retinal thickness +/-SD decreased from 535 +/- 148 microm at baseline to 323 +/- 116 microm at the 6-month follow-up. Ischemic CRVO was associated with significantly lower visual acuity than nonischemic CRVO (P0.001). However, visual acuity gain was similar in both groups. Independent of duration of symptoms, CRVO was associated with a similar gain in visual acuity.Intravitreal injection of bevacizumab appears to be a new treatment option for patients with macular edema secondary to CRVO.

Purpose: To evaluate the advantages, disadvantages, safety, and surgical applicability of the pulsed electron avalanche knife (PEAK-fc), a new electrosurgical knife for “cold” and tractionless cutting, in vitreoretinal surgery. PEAK-fc is equipped with an integrated fiberoptic that makes bimanual procedures in intraocular surgery possible. Methods: A prospective consecutive trial of 18 eyes in 18 patients who underwent vitreoretinal surgery for proliferative diabetic retinopathy, proliferative vitreoretinopathy, subretinal macular hemorrhage, or macular pucker was performed. The following specific maneuvers were performed with PEAK-fc: transection of epiretinal membranes, retinotomies, retinal vessel coagulation, and posterior membranectomy. Results: Detached and attached retina could be dissected successfully in eight cases. Intraoperatively, incision edges were sharply demarcated, showing no visible collateral damage. Deeper layers than the neurosensory retina were not affected. With the bimanual approach, epiretinal avascular and vascular membranes could be removed in 10 cases. Hemorrhages occurring during transection of vascularized membranes could be stopped immediately using the coagulation mode of PEAK-fc. Posterior capsule fibrosis was successfully excised in one patient. No complications were observed. Conclusion: PEAK-fc offers precise and tractionless tissue cutting during ocular surgery. Using different waveform parameters, the same device performs cold cutting and/or “hot” coagulation, thus improving the precision, safety, and ergonomics of vitreoretinal surgery.

Purpose To investigate transglutaminases (enzymes capable of cross-linking extracellular matrix proteins to proteolysis-resistant complexes during scar tissue formation) in a human donor cornea after successful laser in situ keratomileusis (LASIK) without clinical complications and to compare with the results in a human donor cornea with corneal scaring after corneal injury. Setting Department of Ophthalmology, Ludwig-Maximilians University, Munich, Germany. Methods A donor cornea with prior uneventful LASIK treatment and 1 with corneal scaring after penetrating injury were investigated. Cryostat sections were stained immunohistochemically for tissue transglutaminase (tTG), keratocyte transglutaminase (kTG), and their reaction product ɛ-(γ-glutamyl)-lysine. Results With light microscopy, the flap interface of the LASIK-treated eye could hardly be detected, while in the injured eye, infiltration of cells and a clear margin next to the scar formation were present. Immunohistochemistry demonstrated a distinct staining for tTG, kTG, and ɛ-(γ-glutamyl)-lysine in the corneal scar. In contrast, neither transglutaminase nor ɛ-(γ-glutamyl)-lysine staining could be observed at the flap margin or in the interface of the LASIK-treated donor eye. Conclusions Irreversible protein cross-linking of transglutaminases via ɛ-(γ-glutamyl)-lysine connections seem to be indicators for scaring in corneal wound healing. The absence of transglutaminases and their reaction product ɛ-(γ-glutamyl)-lysine in a LASIK-treated cornea supports the idea of missing scar tissue formation after LASIK surgery.

Purpose: To establish an in vitro model of laser in situ keratomileusis (LASIK) in human donor eyes and to test its validity in comparison with animal models. Setting: Department of Anatomy, Friedrich-Alexander Unviersity, Erlangen, Germany. Methods: Laser in situ keratomileusis was performed on 20 organ-cultured human corneal buttons. The excimer laser ablations ranged from 0 to 12.0 diopters. The corneas were maintained in culture for up to 6 months and then evaluated with light microscopy and transmission electron microscopy. In addition, corneal sections were immunohistochemically stained for collagen type III, laminin, and fibronectin. The main outcome measures were the ultrastructural and immunohistochemical features of the stromal incision interface. Results: Ultrastructural investigations in the peripheral cornea revealed a disarrangement of collagen fibers, indicating scar formation. These findings were not observed in the central area. Immunohistochemical staining for fibronectin and collagen type III was detected over the entire stromal incision interface, whereas laminin staining was related to the ingrowth of epithelial cells. Conclusions: The morphological changes after LASIK in an organ culture model can simulate the in vivo situation. Therefore, this model appears appropriate to use in further study of corneal wound-healing changes after LASIK.

Retinal pigment epithelium (RPE) builds the outer blood–retinal barrier of the eye and plays an important role in pathogenesis of the sight threatening disease equine recurrent uveitis (ERU). ERU is a spontaneous autoimmune mediated inflammatory disease characterised by the breakdown of the outer blood–retinal barrier and an influx of autoaggressive T-cells into the inner eye. Therefore, identification of molecular mechanisms contributing to changed function of blood–retinal barrier in ERU is important for the understanding of pathophysiology. Cell surface proteins of RPE collected from healthy horses and horses with ERU were captured by in situ biotinylation and analysed with high resolution mass spectrometry coupled to liquid chromatography (LC–MS/MS) to identify differentially expressed proteins. With label free differential proteomics, a total of 27 differently expressed cell surface proteins in diseased RPE could be detected. Significant down-regulation of three very interesting proteins, synaptotagmin 1, basigin and collectrin was verified and further characterised. Biological significance We applied an innovative and successful method to detect changes in the plasma cell surface proteome of RPE cells in a spontaneous inflammatory eye disease, serving as a valuable model for human autoimmune uveitis. We were able to identify 27 differentially expressed plasma cell membrane proteins, including synaptotagmin 1, basigin and collectrin, which play important roles in cell adhesion, transport and cell communication.

We report the case of a patient presenting with serous retinal detachment following radial optic neurotomy for central retinal vein occlusion. Initially, the retinal detachment was successfully treated by a second vitrectomy and laser coagulation. After reabsorption of the gas tamponade, a recurrence of the retinal detachment was seen with no detectable retinal break. Although other mechanisms should be taken into account, this case indicates that it is possible to create a fistula between the subarachnoid and subretinal space. We conclude from this observation that special attention should be paid to the depth of the radial incision into the optic nerve head.

Purpose To analyze the changes in optic surface roughness before and after injection of various intraocular lens (IOL) models using atomic force microscopy (AFM). Settings Departments of Ophthalmology, Medical University of Graz, General Hospital Linz and University Hospital Basel; Upper Austria University, School of Applied Health and Social Sciences, Linz, Austria. Design Experimental study. Methods The morphology and surface roughness of 3 hydrophobic acrylic IOLs from different manufacturers were analyzed by AFM in liquid using the tapping mode. First, AFM was performed on IOLs taken from the original package without further manipulation. In a second step, under sterile conditions, an experienced cataract surgeon loaded the IOLs into the appropriate injection system and pushed them through a system resembling an IOL implantation in cataract surgery; this was followed by AFM evaluation. Finally, 3 samples of a preloaded hydrophilic acrylic IOL taken from the original cartridge were compared with 3 samples that were pushed through the implantation system. Results Comparison of the arithmetic mean, standard deviation, root mean square, and surface skewness of the IOLs before and after injection showed a significant increase in surface roughness (P Conclusions Standard application procedures of IOLs may alter the IOL surface. Increases in the surface roughness of IOLs may influence postoperative posterior capsule opacification. Further studies are necessary to evaluate the interfacial properties of IOLs. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned.

Background Cumulative light exposure is significantly associated with progression of age-related macular degeneration (AMD). Inhibition of vascular endothelial growth factor is the main target of current antiangiogenic treatment strategies in AMD. However, other growth factors, such as platelet-derived growth factor (PDGF) and placenta growth factor (PlGF), have a substantial impact on development of AMD. Previous reports indicate that sorafenib, an oral multikinase inhibitor, might have beneficial effects on exudative AMD. This study investigates the effects of sorafenib on lightinduced overexpression of growth factors in human retinal pigment epithelial (RPE) cells. Methods Primary human RPE cells were exposed to white light and incubated with sorafenib. Viability, expression, and secretion of VEGF-A, PDGF-BB, and PlGF and their mRNA were determined by reverse transcription-polymerase chain reactions, immunohistochemistry and enzyme-linked immunosorbent assays. Results Light exposure decreased cell viability and increased expression and secretion of VEGF-A, PDGF-BB and PlGF. These light-induced effects were significantly reduced when cells were treated with sorafenib at a dose of 1 mg/ml. Conclusion The results show that sorafenib has promising properties as a potential antiangiogenic treatment for AMD.

The fourth-generation fluoroquinolone, moxifloxacin, covers most gram-positive and gram-negative isolates causing endophthalmitis. It is safe and effective for systemic and topical use, but only limited data are available on prophylactic intracameral administration to prevent endophthalmitis. This study uses a cell culture model to investigate the safety of moxifloxacin for intracameral application.Endothelial toxicity of moxifloxacin was evaluated in cultured human corneas. Possible toxic effects of moxifloxacin (10-750 microg/mL) in corneal endothelial cells (CEC), primary human trabecular meshwork cells (TMC), and primary human retinal pigment epithelial (RPE) cells were evaluated after 24 hours and under conditions of oxidative and inflammatory stress by treatment with tumor necrosis factor alpha, lipopolysaccharides, or interleukin-6. Toxicity was evaluated by tetrazolium dye reduction assay, and cell viability was quantified by a microscopic live-dead assay.No corneal endothelial toxicity could be detected after 30 days of treatment with 500 microg/mL moxifloxacin. Concentrations up to 150 microg/mL had no influence on CEC, TMC, or RPE cell proliferation or on cell viability when administered for 24 hours. After preincubation with tumor necrosis factor alpha, lipopolysaccharides, or interleukin-6 for 24 hours and subsequent treatment with moxifloxacin at concentrations from 10 to 150 microg/mL for 24 hours, no significant decrease in proliferation or viability was observed. Hydrogen peroxide exposure did not increase cellular toxicity.This study showed no significant toxicity for moxifloxacin on CEC, TMC, RPE cells, or human corneal endothelium for concentrations up to 150 microg/mL. The minimum inhibitory concentration of moxifloxacin to inhibit 90% of pathogens commonly encountered in endophthalmitis is known to be in the range of 0.25-2.5 microg/mL. Therefore, prophylactic intracameral use of moxifloxacin at concentrations up to 150 microg/mL may be safely used to prevent endophthalmitis after intraocular surgery.

Purpose To evaluate a capsular tension ring (CTR)–supported anterior and posterior capsule opacification (PCO) model in cadaver eyes. The effect of CTR designs on lens capsule shape and lens epithelial cell (LEC) growth were investigated in vitro. Setting Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. Methods Following open-sky extracapsular cataract extraction, CTR models were implanted in 32 eyes of 16 human donors. The lens capsule expansion by the CTRs was evaluated. The capsular bags supported by the CTRs were excised and maintained at physiological conditions for up to 3 months. The area of LEC coverage over the posterior capsule surface was objectively determined twice a day using a graticule. Results After CTR implantation, all lens capsules could be safely excised and transferred into organ culture. The CTR designs resulted in different shapes of lens capsule expansion. Complete LEC confluence occurred after a mean of 8.25 days ± 2.87 (SD) with the AcriRing KR10 (AcriTec), 6.50 ± 1.0 days with the Acrimed, 8.62 ± 3.34 days with the InjectoRing (Corneal), 9.00 ± 1.87 days with the Morcher 14C, 9.33 ± 0.75 days with the Morcher 2A, and 6.25 ± 0.5 days with the Ophthalmic Innovation CTR. Conclusion The CTR-supported in vitro PCO model offers a physiological method to support the lens capsule and is a reproducible system for the study of LEC proliferation.

Purpose: To evaluate the effect of intravitreal bevacizumab (Avastin) injections on visual acuity (VA) and foveal retinal thickness in patients with macular oedema secondary to branch retinal vein occlusion. Methods: A prospective, non-comparative, consecutive, interventional case series of 34 patients. Patients received repeated intravitreal injections of 1.25 mg bevacizumab. Main outcome measures were VA (Snellen charts and ETDRS) and retinal thickness (optical coherence tomography measurements) in a follow-up period of 6 months. Results: Patients presented at a mean age of 69 years (range 44–86). Mean duration of symptoms was 40 weeks (range 1–300). Mean (SD) VA at baseline was 0.79 (0.39) logMAR, improving to 0.51 (0.34) logMAR at 6 months (p = 0.009). Mean number of letters on the ETDRS chart at baseline was 45.3 (19.0), improving to 60.6 (19.9) at 6 months (p = 0.003). Mean (SD) retinal thickness at baseline was 474 (120) μm, declining to 316 (41) μm at 6 months. Conclusion: Intravitreal injection of 1.25 mg bevacizumb appears to be an effective treatment option for branch retinal vein occlusion.

Background: The pulsed electron avalanche knife (PEAK-fc) is a new pulsed electrosurgical device that allows for precise, “cold” and traction-free tissue dissection. Aim: To evaluate the surgical applicability, safety and potential complications of PEAK-fc in complicated cataract surgery. Methods: The study included five children with congenital cataracts, two patients with advanced senile cataracts, six adults with mature cataracts, three of them with posterior iris synechia, three patients with post-traumatic cataracts with zonulolysis, one patient with intumescent traumatic cataract and three patients with massive anterior capsule opacification. Anterior and posterior capsulotomies, iris synechiolysis, dissection of anterior capsule opacification and fibrotic scar tissue were performed. PEAK-fc was set at voltages of 500–700 V, pulse duration of 0.1 m and repetition rate of 40–100 Hz. Results: Anterior and posterior capsulotomies were successfully and safely performed in all eyes. The edges of capsulotomies appeared sharp, showing only limited collateral damage. PEAK-fc worked best by just gently touching the capsule, thereby avoiding tractional forces or pressure on the lens capsule. Posterior iris synechiae could be released and anterior capsule opacification was dissected without complications. Conclusions: PEAK-fc is a very helpful cutting device for complicated cases of cataract surgery, especially for mature and congenital cataracts, traumatic zonulolysis or anterior segment complications after intraocular inflammation.

PURPOSE. Scarring of the filtering bleb site is the main cause of failure in glaucoma filtration surgery. In the present study, the role of tissue transglutaminase (tTgase) in the accumulation of extracellular matrix (ECM) proteins in these scars was investigated. Transglutaminases are enzymes capable of cross-linking ECM proteins to proteolysis-resistant complexes. METHODS. Expression of tTgase, its reaction product -(-glutamyl)-lysine, and fibronectin and their colocalization were investigated immunohistochemically in failed blebs and in an in vitro trabeculectomy model. Failed blebs were analyzed by RT-PCR for the presence of tTgase mRNA. Human Tenon fibroblasts (HTFs) were treated with transforming growth factor-2 (TGF-2). The effect was studied with immunohistochemistry, Northern blot analysis, and Western blot analysis. tTgase activity was assayed by incorporation of biotinylated cadaverine into fibronectin. RESULTS. Expression of tTgase and -(-glutamyl)-lysine was present in all failed blebs. Staining was most prominent at the rim of the Tenon cyst. In the in vitro trabeculectomy model, tTgase and -(-glutamyl)-lysine were barely present at the incision side of the flap but were perspicuously increased by TGF-2 treatment. Enzyme and its reaction product were colocalized with fibronectin. Cultured HTFs contained a basal level of tTgase mRNA. After treatment with TGF-2, expression and activity of tTgase significantly increased. CONCLUSIONS. The findings demonstrated that tTgase is present and functionally active in failed blebs. Expression and activity of tTgase appeared to be stimulated by TGF-2, a growth factor known to be increased in primary open angle glaucoma. Intervention at this pathway might open a new approach to prevent scarring after glaucoma filtration surgery. (Invest Ophthalmol

Purpose: To compare immunohistochemical findings in human donor corneas after successful laser in situ keratomileusis (LASIK) without clinical complications with a recently established human LASIK in vitro model. Methods: Donor corneas with prior LASIK treatment were investigated. Cryostat sections were stained immunohistochemically for collagen types I, III, and VI and laminin and fibronectin. Results: With light microscopy, the interface of the LASIK flap could hardly be detected. In all samples, fibronectin was consistently detected along the entire extent of the surgical wound. In contrast, collagen type III and laminin only stained the superficial portion of the LASIK incision site. Staining for collagen types I and VI showed no changes after LASIK. Conclusion: Histologic findings in donor corneas with prior LASIK treatment confirm histologic observations in a recently introduced human organ culture LASIK model. This strengthens the reliability of the latter LASIK model for further studies concerning wound healing after LASIK surgery.

Functional research of retinal pigment epithelium (RPE) most often relies on utilization of RPE-derived cell lines in vitro. However, no studies about similarities and differences of the respective cell lines exist so far. Thus, we here analyze the proteome of the most popular RPE cell lines: ARPE-19 and hTERT and compare their constitutive and de novo synthesized protein expression profiles to human early passage retinal pigment epithelial cells (epRPE) by 2-D electrophoresis and MALDI-TOF peptide mass fingerprinting. In all three cell lines the baseline protein expression pattern corresponded well to the de novo synthesized cellular proteome. However, comparison of the protein profile of epRPE cells with that of hTERT-RPE cells revealed a higher abundance of proteins related to cell migration, adhesion, and extracellular matrix formation, paralleled by a down-regulation of proteins attributed to cell polarization, and showed an altered expression of detoxification enzymes in hTERT-RPE. ARPE-19 cells, however, exhibited a higher abundance of components of the microtubule cytoskeleton and differences in expression of proteins related to proliferation and cell death. epRPE cells, hTERT-RPE, and ARPE-19 therefore may respond differently with respect to certain functional properties, a finding that should prove valuable for future in vitro studies.

The pulsed electron avalanche knife (PEAK-fc, Carl Zeiss Meditec) is an electrosurgical cutting device that allows precise "cold" and traction-free tissue dissection. We describe its applicability and safety for anterior capsulotomy in a child with congenital cataract and an adult patient with mature cataract. The PEAK-fc was set at a voltage of 600 V and a pulse repetition rate of 80 Hz. Anterior capsulotomies were successfully and safely performed in both cases, with the edges of capsulotomies appearing sharp and showing only limited collateral damage. The PEAK-fc appears to be a helpful cutting device for complicated cases of cataract surgery, especially for mature and congenital cataracts.

Purpose To determine whether the beta-galactoside-binding matricellular protein Gal-1 is expressed in human specimens of proliferative vitreoretinopathy (PVR) and to evaluate its influence on RPE migration. Methods RT-PCR was used to detect Gal-1-specific transcripts in PVR membranes, and the expression pattern of Gal-1 was examined by immunohistochemistry. Expression of Gal-1 in native, low- and high-density cultured RPE cells was determined by Western blot analysis. Cultured human RPE cells were treated with bFGF, TGF-beta2, PDGF-BB, or HGF. The dose-response of Gal-1 mRNA expression was measured by by real-time quantitative RT-PCR and Northern blot analysis. Induction of Gal-1 protein was confirmed by Western blot analysis. To study the effect of Gal-1 on RPE migration in vitro, Gal-1 expression was silenced by RNA interference. beta-Lactose was used to saturate extracellular galectins. RPE cell migration was assessed by a modified Boyden chamber assay, with HGF as the chemoattractant. Results Gal-1 mRNA expression was present in human specimens of PVR membranes, and staining for Gal-1 was distributed throughout the extracellular matrix (ECM) of PVR membranes. Colocalization was found with laminin and fibronectin and cells of epithelial origin. Western blot analysis revealed greater baseline expression levels in low-density cultured RPE cells than in native and high-density cultured RPE cells. Treatment with HGF caused a dose-dependent increase in Gal-1 expression. Low expression levels of Gal-1 correlated with a reduction of RPE migration to 14% of control. beta-Lactose inhibited HGF-induced RPE cell migration to 23% of control. Conclusions Gal-1 is present in the extracellular matrix of PVR membranes and may be derived from dedifferentiated RPE cells. The expression level of Gal-1 appears to be related to a migratory RPE phenotype and stimulation by HGF, both conditions implicated in the pathogenesis of early PVR. Furthermore, HGF-induced RPE migration may be dependent, at least in part, on Gal-1- and beta-galactoside-dependent mechanisms.

PURPOSE. To evaluate systematically the staining characteristics and safety of potential new dyes for intraocular surgery. METHODS. Six dyes were included in the investigation: light green SF (LGSF) yellowish, E68, bromophenol blue (BPB), Chicago blue (CB), rhodamine 6G, rhodulinblau-basic 3 (RDB-B3). All dyes were dissolved and diluted in a balanced saline saline solution. The light-absorbing properties of each dye were measured at a concentration of 0.05% between 200 and 1000 nm. Staining characteristics were examined by staining lens capsule tissue and epiretinal membranes (ERMs), removed intraoperatively, with dye concentrations of 1.0%, 0.5%, 0.2%, and 0.05%. Enucleated porcine eyes (postmortem time, 9 hours) were also stained. Dye-related toxicity was evaluated by a colorimetric test (MTT) measuring the inhibition of retinal pigment epithelium (RPE) cell proliferation (RAPE-19 and primary human RPE cells, passages 3-6). Cell viability was also quantified based on a two-color fluorescence cell-viability assay. Dyes were investigated in concentrations of 0.2% and 0.02%. RESULTS. All dyes investigated in this study stained human lens capsules, removed intraoperatively; ERMs, peeled during macular pucker surgery; and enucleated porcine eyes, depending on the concentration applied. The long-wavelength absorption maximum of the dyes was within the range of 527 to 655 nm at concentrations of 0.05%. Rhodamine G6 and RDB-B3 showed adverse effects on ARPE-19 cell proliferation at a concentration of 0.2% and were excluded from further investigation in primary RPE cells. The remaining four dyes showed no toxic effect on ARPE-19 and primary RPE cell proliferation at concentrations of 0.2% and 0.02%. Cell viability was affected by LGSF yellowish (0.2%) and CB (0.2% and 0.02%). Two dyes (E68 and BPB) showed no relevant toxicity in vitro. CONCLUSIONS. The systematic evaluation of dyes for intraocular use seems mandatory. In this study four dyes were identified with effective staining characteristics, with two of these dyes having no detectable toxic effect on RPE cells in vitro.

To evaluate a novel technique for three-dimensional mapping of the retinal pigment epithelium (RPE) layer in patients with subfoveal choroidal neovascularization (CNV) due to age-related macular degeneration. Scanning with a recent generation retinal thickness analyzer (RTA) was performed in consecutive patients undergoing fluorescein angiography. From a 3×3mm area centered on the fovea, three-dimensional area maps of the RPE level were calculated by external spreadsheet software. Included were 18 eyes with classic CNV, 18 eyes with occult CNV and 18 eyes from age-matched normal subjects. Repeatability was assessed by measuring 17 eyes with CNV 3 times. In ten additional patients, RTA imaging results were compared with cross-sections obtained by optical coherence tomography. By both methods, distinctive changes in RPE level maps were observed in classic and occult CNV. In classic CNV with the lesion extending over the RPE, only focal irregularities in the anteriorly displaced RPE surface were observed. In contrast, mapping of occult CNV showed a more irregular displacement of the RPE layer. The RPE map standard deviation indicating surface irregularity differed statistically significantly between the groups, with coefficients of variance of 5.9% for controls, 6.1% for classic and 8.8% for occult CNV (P

To evaluate functional outcome after the intraoperative application of 0.06% trypan blue during vitrectomy for macular pucker.Prospective, randomized, comparative study.Forty-three eyes of 43 consecutive patients, 30 women and 13 men, were incorporated in the study. Patients were randomized into two groups: group 1 (n = 22) with trypan blue (0.06%) staining, and group 2 (n = 21) without trypan blue staining. Functional outcome (best-corrected visual acuity, Goldmann perimetry) was evaluated 1 day before surgery, at 6 weeks, and at intervals of 3, 6, and 12 or more months postoperatively. Only patients with an idiopathic macular pucker were included. In all patients, a standard three-port pars plana vitrectomy with peeling of an epiretinal membrane (ERM) and the internal limiting membrane was performed. No other modification of the surgical procedure, except the use of trypan blue was made between the two groups.Mean age was 68.9 years in group 1 (with trypan blue) and 69.4 in group 2 (without trypan blue). Median best-corrected visual acuity was 20/63 in both groups (range, 20/200-20/40; P.5) before surgery. Mean follow-up time was 5.8 months in group 1 and 5.3 months in group 2. After surgery, median visual acuity had increased to 20/40 in both groups (range, 20/500 to 20/20 in group 1 and 20/100 to 20/20 in group 2; P.001 for both groups). The difference between both groups was not statistically significant (P.5). An improvement of visual acuity (gain of 2 or more lines) was seen in 16 patients of both groups. No postoperative visual field defects were noted.Trypan blue-assisted vitrectomy for macular pucker leads to good functional results with no dye-related adverse effects after short follow-up. Trypan blue might be especially applicable in cases in which the borders of the ERM are difficult to define. Hypothetical advantages, such as fewer recurrences of ERMs after trypan blue staining, will have to be evaluated during further follow-up of patients.

PURPOSE Migration and adhesion of dislocated retinal pigment epithelial (RPE) cells to a fibronectin-rich extracellular matrix is an initial step in proliferative vitreoretinopathy (PVR). In the present study, the functional role of cell surface tissue transglutaminase (tTG) in adhesion and migration of RPE cells on fibronectin (Fn) and collagen type I (Col I) after stimulation with TGF-beta2 was investigated. METHODS Cultured human RPE cells were treated with 1.0 ng/mL TGF-beta2 for 24 hours. Cell surface tTG expression was determined by cell fraction analysis. Attachment on Col I, full-length Fn, and its 45-kDa gelatin-binding and 110-kDa cell-binding fragment was measured with an MTT assay. Migration of RPE cells was measured by a Boyden chamber assay, and cell spreading was determined. Experiments were performed in the presence or absence of anti-tTG antibodies and anti-integrin alpha5 and beta1 antibodies. RESULTS TGF-beta2 markedly induced expression of cell-surface tTG on RPE cells and increased attachment and migration on Fn and Col I. Blocking cell surface tTG inhibited attachment, migration, and spreading on Fn and its 45-kDa gelatin-binding fragment, whereas no effect was seen on Col I and the 110-kDa cell-binding Fn fragment. In contrast, blocking of integrin alpha5 and beta1 suppressed adhesion and migration on full-length Fn and the 110-kDa Fn fragment. CONCLUSIONS These data demonstrate that TGF-beta2 increases expression of cell surface tTG, which in turn strengthens adhesion, migration, and spreading of RPE cells on Fn through the 45-kDa gelatin-binding Fn fragment. At the onset of PVR, this mechanism may help RPE cells to attach and migrate on Fn-containing matrices.

OBJECTIVE—To compare the effectiveness of tele-screening using a novel enhanced retinal thickness analyzer (RTA) with onsite routine ophthalmologic examination for diabetic retinopathy. RESEARCH DESIGN AND METHODS—A consecutive series of 31 eyes from diabetic patients were included. All underwent ophthalmologic examination, including stereoscopic dilated funduscopy and scanning with the RTA. The RTA reports consisted of a wide-angle, red-free fundus photograph and a macular-region retinal thickness map. Reports were graded by three independent graders in a masked manner. The diagnoses of proliferative retinopathy, macular edema, and treatment decisions made by the RTA graders and the clinical examiner were compared. RESULTS—On clinical examination 5 of 31 eyes were diagnosed with proliferative diabetic retinopathy (PDR). All five were referred for treatment by two graders and four eyes by one grader. All eyes with PDR and 12 of the 26 eyes with nonproliferative diabetic retinopathy showed severe macular edema. Seven of the 12 eyes with macular edema were clinically eligible for focal laser treatment, and all of them were detected by all RTA graders. Macular thickening was detected in eight eyes by RTA where no treatment was necessary, as judged by clinical examination. Thus, sensitivity was 93% (mean) for detecting PDR and 100% for detecting macular edema, with a specificity of 58–96% depending on the grader. The RTA did not allow valid assessment due to poor image quality in only one case. CONCLUSIONS—Screening for diabetic retinopathy with a combination of wide-angle fundus photography and macular thickness mapping by an objective method, such as optical coherence tomography or the RTA, offers the prerequisites for establishing a successful tele-screening program.

PURPOSE. SNPs in chromosomal region 10q26 harboring PLEKHA1, ARMS2, and Htra1 showed the strongest association with age-related macular degeneration. Recent evidence suggests that in patients homozygous for the risk allele, the lack of synthesis of the poorly characterized ARMS2 is causative of this disorder. The present study was undertaken to gain an understanding of the genuine (patho)physiological role of this protein. METHODS. ARMS2-interacting proteins were identified by using a yeast two-hybrid system and validated by coprecipitation. Immunofluorescence was applied to reveal the localization of ARMS2 in transfected cells and in human eyes. Western blot analyses were performed on extra- and intracellular fractions of ARMS2-expressing cells to demonstrate the secretion of ARMS2. RESULTS. Contrary to previous reports, this study showed that ARMS2 is a secreted protein that binds several matrix proteins. Notably, ARMS2 directly interacts with fibulin-6 (hemicentin1). Mutations in the fibulin-6 gene have been demonstrated to cause familial AMD. ARMS2 also interacts with further extracellular proteins, several of which have been implicated in macular dystrophies. Although ARMS2 apparently lacks any classic targeting sequence, it is translocated to the endoplasmic reticulum in cultured cells before secretion. ARMS2 is mostly confined to choroid pillars in human eyes, representing a part of extracellular matrix and corresponding to the principal sites of drusen formation. CONCLUSIONS. The pivotal role of the extracellular matrix in the progression of AMD is underlined by the abnormal deposition of extracellular debris in the macula, observed frequently in affected individuals. The results have shown that ARMS2 may be necessary for proper matrix function. (Invest Ophthalmol Vis Sci. 2010;51:79‐88) DOI:10.1167/iovs.09-3850

PURPOSE. In early exudative age-related macular degeneration (AMD), segmental thinning of Bruch’s membrane is associated with ingrowth of choroidal neovascularization into the subretinal space. To determine whether there is a link between oxidative stress and extracellular matrix (ECM) degradation by the retinal pigment epithelium, the present study focused on the effect of oxidative stress on MMP-1 and MMP-3 expression, two enzymes with substrate specificity for components of Bruch’s membrane. METHODS. Cultured human RPE cells were exposed to oxidative stress. To investigate the role of signal transduction proteins, cells were pretreated with the specific inhibitors SB202190 or PD98059. Secreted MMP-1 and MMP-3 were detected by ELISA, MMP-2, and MMP-9 by zymography. Expression of mRNA was determined by quantitative real-time RT-PCR. ECM degradation by retinal pigment epithelium was assessed by immunofluorescence microscopy. RESULTS. Oxidative stress increased MMP-1 and MMP-3 protein release but reduced MMP-2 activity. Real-time RT-PCR disclosed increases of MMP-1 and MMP-3 mRNA after oxidative stress with no modulation of TIMP-1. MMP-2 and MMP-9 mRNA was slightly enhanced. PD98059, an inhibitor of ERK1/2, markedly reduced MMP-1 expression, whereas SB202190, an inhibitor of p38 MAPK, was less effective. MMP-3 expression was attenuated by both inhibitors. Oxidative stress–stimulated type I collagen degradation by RPE cells was reduced by simultaneous treatment with a synthetic MMP-inhibitor or a neutralizing antibody against MMP-1. CONCLUSIONS. MMP-1 and MMP-3 in the retinal pigment epithelium are inducible by oxidative stress. The directional shift in the MMP-1,-3/TIMP-1 ratio is associated with increased type I collagen degradation. This may be an important mechanism contributing to the pathogenesis of early exudative AMD. (Invest Ophthalmol Vis Sci. 2009;50:5495–5503) DOI:10.1167/ iovs.08-3193

PURPOSE Proliferative vitreoretinopathy (PVR) is an excessive wound-healing process and the major complication in rhegmatogenous retinal detachment. The present study was designed to investigate the expression of keratinocyte transglutaminase (kTgase) in PVR membranes and retinal pigment epithelial (RPE) cells and to evaluate its expression in response to growth factors known to be increased in PVR disease. METHODS Distribution of kTgase and its relation to fibronectin have been investigated immunohistochemically. PVR membranes and native and cultured RPE cells were analyzed by RT-PCR for the presence of kTgase mRNA. In vitro, RPE cells were treated with transforming growth factor (TGF)-beta2, basic fibroblast growth factor, interleukin-6, and interleukin-1beta. Expression of kTgase was studied by Northern and Western blot analysis. The effect of connective tissue growth factor (CTGF) silencing on the TGF-beta2-modulated expression of kTgase was investigated by transfection with CTGF small interfering (si)RNA before TGF-beta2 treatment. RESULTS mRNA expression of kTgase was present in all PVR membranes. Immunohistochemical staining for kTgase showed an inhomogeneous pattern with localized accumulation and little colocalization with fibronectin. Although native RPE cells contained only a basal level of kTgase mRNA, the expression of kTgase was increased under culture conditions and was further enhanced by TGF-beta2 treatment. Silencing of CTGF expression did not attenuate the TGF-beta2-mediated induction of kTgase. CONCLUSIONS The findings demonstrate that kTgase is present in PVR membranes. Its amount is related to the differentiation state of RPE cells and stimulation by TGF-beta2. TGF-beta2-mediated increase seems to be independent of CTGF.

Objective To evaluate the effectiveness and precision of tractionless retinal tissue dissection by the advanced version of the pulsed electron avalanche knife for fine cutting (PEAK-fc; Carl Zeiss Meditec, Jena, Germany). Methods Porcine retina (in vivo) and human retina (in vitro) were incised with the PEAK-fc using various pulse parameters. The globes were then processed for light microscopy. Evaluation of all specimens focused on depth of the retinal cuts and on the degree of collateral damage. Results Retinal cuts performed both in vivo on porcine eyes and on human donor eyes showed very sharp edges with only little collateral damage. With probes of 600 μm in length, the optimal pulse parameters for precise and reproducible cutting of the retina were an amplitude of 350 to 380 V, a repetition rate of 300 Hz, and 30 “minipulses” per pulse of 100-microsecond duration. With increasing voltage, cuts also affected the retinal pigment epithelium and the choroid, followed by intravitreal bleeding during in vivo application. Conclusion We demonstrated that PEAK-fc is capable of precisely cutting retinal tissue in vivo and in vitro using optimal pulse parameters. Further in vivo studies will be necessary to determine the efficacy of this new tractionless cutting device in vitreoretinal surgery.

Purpose Dedifferentiation of retinal pigment epithelial (RPE) cells is a crucial event in the pathogenesis of proliferative vitreoretinopathy (PVR). This study was designed to improve the understanding of RPE cell dedifferentiation in vitro. The protein expression pattern of native differentiated RPE cells was compared with that of cultured, thereby dedifferentiated, RPE cells. Methods Differentiated native human RPE cells and monolayers of dedifferentiated cultured primary human RPE cells were processed for two-dimensional (2-D) electrophoresis. Total cellular proteins were separated by isoelectric focusing using immobilized pH gradients (IPG 3-10) and electrophoresis on 9% to 15% gradient polyacrylamide gels. Proteins were visualized by silver staining. Silver-stained gel spots were excised, digested in situ, and analyzed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy (MS). The resultant peptide mass fingerprints were searched against the public domain NCBInr, MSDB, and EnsemblC databases to identify the respective proteins. Results One hundred seventy nine protein spots were analyzed and classified into functional categories. Proteins associated with highly specialized functions of the RPE, which are required for interaction with photoreceptor cells, including RPE65, cellular retinaldehyde-binding protein (CRALBP), and cellular retinol-binding protein (CRBP), were absent in dedifferentiated cultured RPE cells, whereas proteins involved in phagocytosis and exocytosis, including cathepsin D and clathrin were still present. Dedifferentiated RPE cells displayed a strong shift toward increased expression of proteins associated with cell shape, cell adhesion, and stress fiber formation, including cytokeratin 19, gelsolin, and tropomyosins, and also acquired increased expression of factors involved in translation and tumorigenic signal transduction such as annexin I and translation initiation factor (eIF)-5A. Conclusions Dedifferentiation of human RPE cells in vitro results in downregulation of proteins associated with highly specialized functions of the RPE and induces the differential expression of proteins related to cytoskeleton organization, cell shape, cell migration, and mediation of proliferative signal transduction. These in vitro data suggest that the dedifferentiated status of RPE cells per se may initiate PVR. Further investigation of candidate proteins may identify additional targets for treatment or prevention of diseases associated with RPE dedifferentiation.

PURPOSE. Proliferative vitreoretinopathy (PVR) is characterized by the development of epi- and subretinal fibrocellular membranes containing modified retinal pigment epithelial (RPE) cells among others. In the present study, the role of transglutaminases in accumulation of extracellular matrix (ECM) proteins in these membranes was investigated. Transglutaminases are enzymes capable of cross-linking ECM proteins to proteolysis-resistant complexes. METHODS. PVR membranes were incubated with dansyl-cadaverine to demonstrate active transglutaminase. Localization of tissue transglutaminase (tTgase), its reaction product -(-glutamyl)-lysine, and fibronectin was investigated immunohistochemically. Colocalization was studied with a confocal laser scanning microscope. PVR membranes were also analyzed by RT-PCR for the presence of tTgase mRNA. In vitro, RPE cells were treated with transforming growth factor-2 (TGF-2), basic fibroblast growth factor, interleukin-6, and interleukin1. Their effect was studied using immunohistochemistry and Northern and Western blot analyses. RESULTS. Transglutaminase activity and expression of tTgase were present in all PVR membranes. Staining was most prominent at the rim of the membranes. The enzyme was colocalized with -(-glutamyl)-lysine and fibronectin. No staining differences were found between epi- and subretinal membranes. Although native RPE cells contained only a basal level of tTgase mRNA, the expression and activity of tTgase was increased under culture conditions and further stimulated by TGF-2 treatment. CONCLUSIONS. The findings demonstrate that in PVR membranes tTgase is present and functionally active. The amount and activity of this enzyme appears to be related to the differentiation state of the RPE cells and their stimulation by TGF-2, a growth factor known to be increased in the vitreous of PVR. Intervention at this pathway may open a new approach for PVR prevention and therapy. (Invest Ophthalmol Vis Sci. 2003; 44:355‐364) DOI:10.1167/iovs.02-0224