Galectin-1 and -3 in high amounts inhibit angiogenic properties of human retinal microvascular endothelial cells in vitro.

PURPOSE: Galectin-1 and -3 are ß-galactoside binding lectins with varying effects on angiogenesis and apoptosis. Since in retinal pigment epithelial cells high amounts of human recombinant galectin (hr-GAL)1 and 3 inhibit cell adhesion, migration and proliferation, we investigated if hr-GAL1 and 3 have homologous effects on human retinal microvascular endothelial cells (HRMEC) in vitro. METHODS: To investigate the effect of galectin-1 and -3 on HRMEC, proliferation, apoptosis and viability were analyzed after incubation with 30, 60 and 120 µg/ml hr-GAL1 or 3 by BrdU-ELISA, histone-DNA complex ELISA, live/dead staining and the WST-1 assay, respectively. Further on, a cell adhesion as well as tube formation assay were performed on galectin-treated HRMEC. Migration was investigated by the scratch migration assay and time-lapse microscopy. In addition, immunohistochemical staining on HRMEC for ß-catenin, galectin-1 and -3 were performed and ß-catenin expression was investigated by western blot analysis. RESULTS: Incubation with hr-GAL1 or 3 lead to a decrease in proliferation, migration, adhesion and tube formation of HRMEC compared to the untreated controls. No toxic effects of hr-GAL1 and 3 on HRMEC were detected. Intriguingly, after treatment of HRMEC with hr-GAL1 or 3, an activation of the proangiogenic Wnt/ß-catenin signaling pathway was observed. However, incubation of HRMEC with hr-GAL1 or 3 drew intracellular galectin-1 and -3 out of the cells, respectively. CONCLUSION: Exogenously added hr-GAL1 or 3 inhibit angiogenic properties of HRMEC in vitro, an effect that might be mediated via a loss of intracellular endogenous galectins.

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

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

Quality of life in the follow-up of uveal melanoma patients after CyberKnife treatment.

To assess quality of life in uveal melanoma patients within the first and second year after CyberKnife radiosurgery. Overall, 91 uveal melanoma patients were evaluated for quality of life through the Short-form (SF-12) Health Survey at baseline and at every follow-up visit over 2 years after CyberKnife radiosurgery. Statistical analysis was carried out using SF Health Outcomes Scoring Software and included subgroup analysis of patients developing secondary glaucoma and of patients maintaining a best corrected visual acuity (BCVA) of the treated eye of 0.5 log(MAR) or better. Analysis of variance, Greenhouse-Geisser correction, Student's t-test, and Fisher's exact test were used to determine statistical significance. Physical Functioning (PF) and Role Physical (RP) showed a significant decrease after CyberKnife radiosurgery, whereas Mental Health (MH) improved (P=0.007, P<0.0001 and P=0.023). MH and Social Functioning (SF) increased significantly (P=0.0003 and 0.026) in the no glaucoma group, MH being higher compared with glaucoma patients (P=0.02). PF and RP were significantly higher in patients with higher BCVA at the second follow-up (P=0.02). RP decreased in patients with BCVA<0.5 log(MAR) (P=0.013). Vitality (VT) increased significantly in patients whose BCVA could be preserved (P=0.031). Neither tumor localization nor size influenced the development of secondary glaucoma or change in BCVA. Although PF and RP decreased over time, MH improved continuously. Prevention of secondary glaucoma has a significant influence on both SF and MH, whereas preservation of BCVA affects VT. Emotional stability throughout follow-up contributes positively toward overall quality of life. CyberKnife radiosurgery may contribute to attenuation of emotional distress in uveal melanoma patients.

Corneal cell response after flap creation using a mechanical microkeratome or a 200 kHz femtosecond laser.

PURPOSE: To compare the inflammatory cell response within the corneal flap interface created by a mechanical microkeratome and a femtosecond laser. SETTING: Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. DESIGN: Experimental in vitro study. METHODS: Corneoscleral buttons of 12 enucleated human eyes not suitable for transplantation were put into organ culture. Corneal flaps were created using a 200 kHz femtosecond laser (Visumax) (femtosecond group) or a mechanical microkeratome (Amadeus) (microkeratome group). Flaps were not lifted after treatment. In 2 corneas, no treatment was performed (control group). Corneas were kept in organ culture for 12 hours thereafter. To evaluate cell-mediated immune reaction, immunofluorescent staining for leucocytes (cluster of differentiation 45) and specifically for dendritic cells (human leukocyte antigen-DR) was performed in every group. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to determine apoptosis reaction. RESULTS: The ratio of dendritic cells in the femtosecond group compared with the microkeratome group was 1.2 (P=.02), the ratio of leucocytes was 1.4 (P=.06), and the ratio of apoptotic cells was 1.0 (P=.59). There was no marked significant difference in the distribution of inflammatory cell reaction. The control group showed neither specific inflammatory reaction nor apoptosis. CONCLUSION: This in vitro series of human corneas showed similar inflammatory tissue reaction after femtosecond laser-assisted and microkeratome-assisted flap creation (P<.05). FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Alkylphosphocholines for intraocular lens coating.

PURPOSE: To assess the effect of alkylphosphocholine (APC)-coated intraocular lenses (IOLs) on human lens epithelial cell (LEC) proliferation in vitro and the corneal biocompatibility of APC in an organ culture model of human donor eyes. SETTING: Research Laboratory for Experimental Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. DESIGN: Experimental study. METHODS: Six foldable IOLs differing in optic material were incubated with APC, washed in phosphate-buffered saline, and dried overnight. Intraocular lenses of the same lot served as uncoated controls. Each rehydrated IOL was placed in 1 well of a 24-well plate containing proliferating human LECs. Cell survival was tested by the tetrazolium-dye reduction assay 5 days later. Biocompatibility of the human corneal endothelium was assessed by a 24-hour incubation of human donor corneas with different concentrations of APC before the live/dead assay was performed. RESULTS: Human LEC proliferation was attenuated by APC-coated IOLs (P=.001). Coated hydrophilic acrylic IOLs were more effective inhibitors of human LEC proliferation than coated hydrophobic acrylic or silicone IOLs (P=.001). Alkylphosphocholines were well tolerated by the corneal endothelium in an organ culture model of human donor corneas up to a concentration of 1 mM (n = 12). CONCLUSIONS: Results show that APCs are suitable agents for IOL coating without linker molecules. Coated IOLs can inhibit human LEC proliferation and were well tolerated by human donor corneas in organ culture.

Synthesis, staining properties, and biocompatibility of a new cyanine dye for ILM peeling.

BACKGROUND: The aim of this work is to investigate the biocompatibility and staining properties of DSS: 3,3'-Di-(4-sulfobutyl)-1,1,1',1'-tetramethyl-di-1H-benz[e]indocarbocyanine (DSS). METHODS: Dye concentrations of 0.5, 0.25, and 0.1% were evaluated (290 and 295 mOsm). Toxicity was assessed using a colorimetric test measuring the inhibition of ARPE 19 cell, human primary RPE cell, and human Müller cell proliferation. Exposure time was 30, 60, 120, and 300 s. Indocyanine green (ICG) (0.5, 0.25, and 0.1%) served as a control. Cells were also illuminated with plain white light (750 mW/cm(2)) for 10 min to assess phototoxic effects. Besides staining of porcine and human lens capsule, internal limiting membrane (ILM)-staining was assessed by applying 0.25 and 0.5% DSS over the macula in two human post-mortem eyes. RESULTS: DSS of 0.25 and 0.1% showed no toxic effect on primary RPE cells and MIO-M1cells, and 0.5, 0.25, and 0.1% for ARPE-19 cells. In MIO-M1cells, 0.5% dye showed a significant reduction of mitochondrial dehydrogenase activity only following an exposure time of 300 s. Following illumination, ICG showed a significantly more pronounced effect on cell viability in primary RPE cells and MIO-M1cells compared to DSS. The absorption maximum is found at 591 nm; the even more bathochromic fluorescence proceeds with a common Stokes' shift where maxima at 620 and 660 nm with a quantum yield of 32% were found. The fluorescence is sufficiently hypsochromic and the fluorescence quantum yield high enough for an easy visual detection. The contrast and staining properties at the ILM were excellent and allowed for a controlled removal of the ILM during surgery. No penetration into deeper retinal layers was noted. CONCLUSIONS: Our results indicate that this new cyanine dye DSS may represent an alternative for ILM staining due to its matched absorption concerning visibility and fluorescence qualities as well as its good biocompatibility.

Axitinib modulates hypoxia-induced blood-retina barrier permeability and expression of growth factors.

This study investigates the effects of the multikinase inhibitor axitinib on the expression of vascular endothelial growth factor (VEGF) receptors 1/2 (VEGFR-1/2) and platelet-derived growth factor (PDGF) receptor beta (PDGFR-ß), hypoxia-induced increased tissue permeability, occludin, zonula occludens protein 1 (ZO-1), VEGF-A, and PDGF expression of human retinal pigment epithelial (RPE) cells and human umbilical vein endothelial cells (HUVECs). Primary human RPE cells and HUVECs were exposed to hypoxia and axitinib. Viability of cells, tissue permeability, and expression of occludin, ZO-1, VEGF, PDGF, VEGFR-1/2 and PDGFR-ß, and their mRNAs, were investigated by reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, western blotting, and immunohistochemistry. Treatment with axitinib reduced expression of VEGFR-1/2 and PDGFR-ß. Hypoxia decreased cell viability, occludin, and ZO-1 expression and increased tissue permeability, expression, and secretion of VEGF and PDGF. Axitinib significantly reduced hypoxia-induced effects on HUVEC and RPE cells. Our in vitro results suggest that axitinib may have promising properties as a potential treatment for diabetic macular edema.

Immunocytochemical and ultrastructural evidence of glial cells and hyalocytes in internal limiting membrane specimens of idiopathic macular holes.

PURPOSE: To provide new information on epiretinal cell proliferation and the cells' origin in idiopathic macular holes and to overcome the effects of embedding and sectioning preparation procedures on cell-distribution patterns. METHODS: Interference and phase-contrast microscopy, immunocytochemistry, and scanning and transmission electron microscopy were performed on surgically excised whole-mounted internal limiting membrane (ILM) specimens removed from 60 eyes with idiopathic macular holes. Cell distribution and cell morphology were correlated with immunocytochemical staining characteristics. Twelve cell type-specific antibodies were used to detect glial cells, hyalocytes, retinal pigment epithelial cells, retinal ganglion cells, and immune cells. Cell viability was analyzed. RESULTS: Epiretinal cell proliferation was found in all ILM specimens, irrespective of the stage of the macular hole. Cell density showed a broad variety. Immunocytochemistry frequently revealed simultaneous expression of GFAP/CD45, GFAP/CD64, GFAP/CD68, GFAP/CRALBP, and GFAP/CD90. Some cells presented with intracellular contractile filaments (anti-αSMA); others were not immunoreactive to any antibody examined. The percentage of viable cells showed a broad variety with a mean of 73% (SD 29%). Electron microscopy demonstrated glial cells, hyalocytes, and myofibroblast-like cells. CONCLUSIONS: The presence of epiretinal cells at the ILM in all macular hole stages strongly suggests a substantial involvement of cell migration and proliferation in the course of macular hole development. Glial cells and hyalocytes play the predominant role in epiretinal cell proliferation. Given the co-expression of glial cell and hyalocyte markers, transdifferentiation of epiretinal cells needs further elucidation, especially with respect to αSMA-positive cells leading to traction at the vitreoretinal interface.

Cyanine dyes as optical contrast agents for ophthalmological surgery.

Cyanine dyes were prepared as optical contrast media for supporting the surgery of the lamina limitans interna (LLI) of the retina and other structures of the human eye. Their absorption spectra were adapted both to the spectral sensitivity of the human eye and to standard illumination. The contrast could be further amplified by the application of the strong fluorescence of the dyes used. The binding of the dyes to various surfaces was studied. No toxic effects could be detected for the applied dyes.

Sequential epiretinal membrane removal with internal limiting membrane peeling in brilliant blue G-assisted macular surgery.

PURPOSE: To assess the selectivity of brilliant blue G (BBG) staining by analysing the morphological components of unstained and stained tissue obtained during epiretinal membrane (ERM) removal with internal limiting membrane (ILM) peeling in BBG-assisted macular surgery. METHODS: Twenty-six surgical specimens were removed from 13 eyes with epiretinal gliosis during vitrectomy using BBG for ERM and ILM peeling. We included eyes with idiopathic macular pucker, idiopathic macular hole and vitreomacular traction syndrome. The dye was injected into the fluid-filled globe. Unstained and stained epiretinal tissue was harvested consecutively and placed into separate containers. All specimens were processed for conventional transmission electron microscopy. RESULTS: The first surgical specimen of all eyes showed no intraoperative staining with BBG and corresponded to masses of cells and collagen. The second surgical specimen demonstrated good staining characteristics and corresponded to the ILM in all patients included. In seven eyes, the ILM specimens were seen with minor cell proliferations such as single cells or a monolayer of cells. Myofibroblasts, fibroblasts and astrocytes were present. In five cases, native vitreous collagen fibrils were found at the ILM. In six of the eyes, ILM specimens were blank. CONCLUSION: Our clinicopathological correlation underlines the selective staining properties of BBG. The residual ILM is selectively stained by BBG even when a small amount of cells and collagen adheres to its vitreal side. To reduce the retinal exposure to the dye, the surgeon might choose to remove the ERM without using the dye, followed by a BBG injection to identify residual ILM.

Minocycline is cytoprotective in human trabecular meshwork cells and optic nerve head astrocytes by increasing expression of XIAP, survivin, and Bcl-2.

INTRODUCTION: Primary open-angle glaucoma (POAG) is one of the leading causes of blindness. Activation of optic nerve head astrocytes (ONHA) and loss of trabecular meshwork cells (TMC) are pathognomonic for this neurodegenerative disease. Oxidative stress and elevated levels of transforming growth factor beta (TGFbeta) play an important role in the pathogenesis of POAG. This study investigates the possible antiapoptotic and cytoprotective effects of minocycline on TMC and ONHA under oxidative stress and increased TGFbeta levels. METHODS: TMC and ONHA were treated with minocycline 1-150 muM. Possible toxic effects and IC(50) were evaluated after 48 hours. Cell proliferation and viability were examined in order to assess the protective effects of minocycline on TMC and ONHA. Expression of Bcl-2, XIAP, and survivin, as well as their mRNA expression, were assessed by real time polymerase chain reaction (RT-PCR) and Western Blot analysis 48 hours after treatment with minocycline alone and additional incubation with TGFbeta-2 or oxidative stress. RESULTS: Minocycline 1-75 muM showed no toxic effects on TMC and ONHA. Under conditions of oxidative stress, both TMC and ONHA showed an increase in viability and an ability to proliferate when treated with minocycline 20-40 muM. RT-PCR and Western blotting yielded an overexpression of Bcl-2, XIAP, and survivin when TMC or ONHA were treated with minocycline 20-40 muM under conditions of oxidative stress and when additionally incubated with TGFbeta-2. CONCLUSION: Minocycline up to 75 muM does not have toxic effects on TMC and ONHA. Treatment with minocycline 20-40 muM led to increased viability and proliferation under oxidative stress and TGFbeta-2, as well as overexpression of Bcl-2, XIAP, and survivin. This protective pathway may help to prevent apoptotic cell death of TMC and ONHA and therefore be a promising approach to avoidance of progression of glaucomatous degeneration.

Experimental evaluation of aniline and methyl blue for intraocular surgery.

PURPOSE: The purpose of this study was to investigate the biocompatibility of aniline and methyl blue in a well-established cell culture model and assess the staining properties of these dyes at the level of the internal limiting membrane (ILM) in human donor eyes. METHODS: Dye-related toxicity was evaluated by a colorimetric test (MTT) measuring the inhibition of retinal pigment epithelium (ARPE-19 and primary human retinal pigment epithelium) cell proliferation. Cell viability was also quantified based on a two-color fluorescence assay (Life-Dead Assay). Aniline blue and methyl blue at a concentration of 0.2% was applied over the macula during vitrectomy in human donor eyes to evaluate the staining properties at the level of the ILM. RESULTS: Both dyes and dye concentrations of 0.1% and 0.2% showed no toxic effect on ARPE-19 and primary human retinal pigment epithelium cell proliferation for exposure times of 1 and 10 minutes, respectively. Cell viability was also not affected at all. Both dyes provided a good contrast at the level of the ILM and allowed for a controlled removal of the ILM during surgery. No penetration into deeper retinal layers was noted. CONCLUSION: Our results indicate that aniline blue and methyl blue might be applicable for intraocular surgery, providing a very good biocompatibility and required selective staining characteristics at the level of the ILM.

Alkylphosphocholines: a new approach to inhibit cell proliferation in proliferative vitreoretinopathy.

Proliferative vitreoretinopathy represents the major complication in retinal detachment surgery and occurs in about 5-15% of cases resulting in a significant loss of vision despite multiple surgical procedures. Although successful anatomical reattachment is usually achieved, the reduction in central vision often remains permanent due to the intraretinal changes induced by retinal detachment and the subsequent proliferative response within the retina. Retinal Muller glial cells play a pivotal role in this process together with retinal pigment epithelial cells which are dispersed in the vitreous and stimulated by growth factors and serum in the vitreous after the breakdown of the blood-retinal barrier.

Intracameral moxifloxacin: in vitro safety on human ocular cells.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Alkylphosphocholines as a potential pharmacologic prophylaxis for posterior capsule opacification.

PURPOSE: To evaluate the effect of alkylphosphocholines (APCs) on human lens epithelial cell (LEC) proliferation, attachment, and migration in a well-established in vitro model. SETTING: Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. METHODS: The immortalized human LEC line HLE-B3 was incubated for 24 hours with APC in different concentrations in the presence of Eagle's modified essential medium supplemented with fetal calf serum under standard cell-culture conditions. The trypan blue exclusion test and live-dead assay were performed to exclude toxic concentrations. To determine cell proliferation, cells were incubated with APCs at the maximum slope of the growth curve for 24 hours before the tetrazolium dye-reduction assay (MTT test) was performed. After cells were seeded on coated 24-well plates, incubated with APCs, and rinsed with phosphate-buffered saline, cell attachment was assessed by the MTT test. Migration was determined by a modified Boyden chamber method after incubation of LECs with APCs. RESULTS: Alkylphosphocholines were effective inhibitors of human LEC proliferation, attachment, and migration at nontoxic concentrations in vitro. The 50% inhibitory concentration was close to 0.1 mM. An APC concentration of 1.0 mM accounted for the following: inhibition of cell proliferation of more than 80%, reduction in cell attachment to 66.5%, and inhibition of cell migration of more than 90%. All effects were dose dependent. No toxic effects were detected compared with controls. CONCLUSIONS: Alkylphosphocholines might have the potential for topical application as a single-dose agent to prevent posterior capsule opacification formation. However, further studies are needed before a clinical application can be considered.

Cytoprotective effects of a blue light-filtering intraocular lens on human retinal pigment epithelium by reducing phototoxic effects on vascular endothelial growth factor-alpha, Bax, and Bcl-2 expression.

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-alpha (VEGF-alpha), 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-alpha 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.

Capsular tension ring-based in vitro capsule opacification model.

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.

The role of alkylphosphocholines in retinal Müller glial cell proliferation.

PURPOSE: Alkylphosphocholines (APCs) are investigated for their effect on Müller glial cell proliferation and F-actin stress fiber distribution in vitro. MATERIALS AND METHODS: Müller cells were incubated with APCs (C18:1-PC and C22:1-PC) +/- fetal calf serum. Proliferation was assessed by BrdU labeling and with the tetrazolium dye-reduction assay. Toxicity was measured using the trypan blue exclusion assay. The distribution of F-actin stress fibers was determined using FITC-phalloidin staining. RESULTS: APCs are effective inhibitors of human and rat Müller glial cell proliferation and hypoxia-induced up-regulation of F-actin stress fibers in vitro in non-toxic concentrations. CONCLUSIONS: APCs might prevent intraretinal changes as a result of serum stimulation and hypoxia following retinal detachment.

The effect of alkylphosphocholines on intraretinal proliferation initiated by experimental retinal detachment.

PURPOSE: To determine the effect of alkylphosphocholines (APCs) on intraretinal proliferation induced by experimental retinal detachment in the rabbit. METHODS: Retinal detachments were created in adult pigmented rabbits. APCs, either liposome bound (liposome, L-APC) or unbound (free, F-APC), were injected intravitreally on either day 1 or day 2 after detachment. BrdU was injected on day 3, 4 hours before death. After fixation, retinas were triple labeled with anti-BrdU, anti-vimentin, and the isolectin B4. The number of anti-BrdU-labeled cells was counted per millimeter of retina from sections imaged by laser scanning confocal microscopy. Toxicity was examined using toluidine blue-stained sections imaged by light microscopy and by electron microscopy for ultrastructural evaluation. RESULTS: Retinal detachment initiated proliferation of all non-neuronal cells. After intravitreal injection on day 1 or 2 after experimental induction of retinal detachment, APCs significantly reduced the number of dividing cells at day 3. Liposome-bound drug given on day 2 was more effective on Müller cell proliferation than was unbound drug. Injection of F-APC on day 1 was more effective than when given on day 2. No apparent effect was seen on Müller cell hypertrophy as indicated by vimentin expression. In addition, no evidence of toxicity was observed in the retina at day 3 for any of the conditions. CONCLUSIONS: APCs significantly reduce the number of Müller cells that are stimulated to divide as a result of retinal detachment. The preliminary results indicate no evidence of significant toxicity; however, further studies are needed. APCs have the potential to be used as part of a therapeutic approach if they can be combined with other agents that can suppress the fibrosis that is also a critical event in the pathogenesis of proliferative vitreoretinal diseases such as proliferative vitreoretinopathy (PVR).

Hypoxia/reoxygenation and TGF-beta increase alphaB-crystallin expression in human optic nerve head astrocytes.

Reactive astrocytes in glaucomatous optic nerve changes are characterized by an increased expression of alphaB-crystallin and transforming growth factor-beta (TGF-beta). In the pathogenesis of glaucomatous optic nerve damage, ischemia/reperfusion injury may play an important role. The goal of the present study was to determine the influence of hypoxia/reoxygenation and TGF-beta on the expression of alphaB-crystallin in cultured human astrocytes of the optic nerve head (ONH). Cultured human astrocytes were incubated under hypoxic conditions (1% O2 for 4-12 h) with subsequent reoxygenation (12-24 h). Additionally, cells were treated with 1.0 ng/ml TGF-beta1 and TGF-beta2 for 12-48 h. Expression of alphaB-crystallin was examined by Northern- and Western-blotting. Levels of TGF-beta1 and TGF-beta2 were analyzed by RT-PCR analysis and ELISA. The effect of TGF-beta blocking on the hypoxia/reoxygenation modulated expression of alphaB-crystallin was investigated by simultaneous incubation with neutralizing antibodies against TGF-beta during the reoxygenation phase. Hypoxia/reoxygenation increased the expression of alphaB-crystallin at the mRNA (2.8- to 3.1-fold) and protein level (1.8- to 2.1-fold). Treatment with 1.0 ng/ml TGF-beta1 and TGF-beta2 for 12-48 h markedly enhanced alphaB-crystallin mRNA expression approximately three- to fourfold. Using Western blot analysis, this increase ranged from 2 to 3 times. Both cytokines showed a twofold increase after 12 and 24 h of reoxygenation at the mRNA and a two- to threefold increase at the protein level. Simultaneous treatment with neutralizing antibodies against both TGF-beta isoforms prevented the hypoxia/reoxygenation-mediated elevation of alphaB-crystallin. The process of hypoxia/reoxygenation is capable of inducing the expression of alphaB-crystallin and TGF-ss in cultured ONH astrocytes. Therefore, optimization of conditions leading to hypoxia/reoxygenation in the ONH of glaucomatous patients may help to lower the incidence of characteristic changes in the optic nerve.