A Comparison of Laboratory and Industrial Processes Reveals the Effect of Dwell Time and UV Pre-Exposure on the Behavior of Two Polymers in a Disintegration Trial.

Biodegradable biopolymers such as polylactic acid and polybutylene succinate are sustainable alternatives to traditional petroleum-based plastics. However, the factors affecting their degradation must be characterized in detail to enable successful utilization. Here we compared the extruder dwell time at three different melt-spinning scales and its influence on the degradation of both polymers. The melt temperature was the same for all three processes, but the shear stress and dwell time were key differences, with the latter being the easiest to measure. Accelerated degradation tests, including quick weathering and disintegration, were used to evaluate the influence of dwell time on the structural, mechanical, and thermal properties of the resulting fibers. We found that longer dwell times accelerated degradation. Quick weathering by UV pre-exposure before the disintegration trial, however, had a more significant effect than dwell time, indicating that degradation studies with virgin material in a laboratory-scale setting only show the theoretical behavior of a product in the laboratory. A weathered fiber from an industrial-scale spinning line more accurately predicts the behavior of a product placed on the market before ending up in the environment. This highlights the importance of optimizing process parameters such as the dwell time to adapt the degradability of biopolymers for specific applications and environmental requirements. By gaining a deeper insight into the relationship between manufacturing processes and fiber degradability, products can be adapted to meet suitable performance criteria for different applications.

Chorioretinal Toxicity of Perfluorooctane (Ala Octa): Results From 48 Surgical Procedures in Geneva.

PURPOSE: To compare outcomes of patient who underwent surgery using perfluorooctane (PFO; C8F18; Ala Octa) with those who underwent surgery with perfluorodecalin (F-Decalin). DESIGN: Retrospective, consecutive, comparative, interventional case series. METHODS: A total of 48 eyes that underwent vitrectomy with PFO were compared to 29 eyes that underwent vitrectomy with perfluorodecalin. Two experienced surgeons performed vitrectomies at the Geneva University Eye Clinic. Visual acuity before, at 8 and 24 weeks after surgery, was documented, and spectral domain optical coherence tomography (SD-OCT) images were analyzed for abnormalities. RESULTS: Two patients experienced severe retinal toxicity, including 1 with severe vision loss. However, no statistical differences in VA were observed between the PFO and perfluorodecalin patients. Analysis of SD-OCT images showed differences in occurrence of several abnormalities, for example, inner segment-outer segment alterations were found in 60.4% of eyes treated with PFO and in 10.3% of perfluorodecalin-treated eyes; retinal atrophic areas were found in 41.7% of PFO and in none of the perfluorodecalin eyes; inner limiting membrane contraction was found in 58.4% of PFO and in none of perfluorodecalin eyes; inner retina cystic alterations were found in 58.3% of PFO eyes and 17.2% of perfluorodecalin eyes; outer retina cystic alterations were found in 39.6% of PFO eyes and 13.8% of perfluorodecalin eyes; retinal holes were found in 14.6% of PFO eyes and in none of the perfluorodecalin eyes; and outer retinal inclusions were found in 20.8% of PFO eyes and in 3.45% of perfluorodecalin eyes. CONCLUSIONS: Perfluorooctane caused significantly more toxic damage than perfluorodecalin. Special consideration should be given to develop a central European Union (EU) control agency for medical devices and to reevaluate safety procedures currently accepted by the EU and International Organization for Standardization for intraocular surgery.

Immunological response to nitroglycerin-loaded shear-responsive liposomes in vitro and in vivo.

Liposomes formulated from the 1,3-diamidophospholipid Pad-PC-Pad are shear-responsive and thus promising nano-containers to specifically release a vasodilator at stenotic arteries. The recommended preclinical safety tests for therapeutic liposomes of nanometer size include the in vitro assessment of complement activation and the evaluation of the associated risk of complement activation-related pseudo-allergy (CARPA) in vivo. For this reason, we measured complement activation by Pad-PC-Pad formulations in human and porcine sera, along with the nanopharmaceutical-mediated cardiopulmonary responses in pigs. The evaluated formulations comprised of Pad-PC-Pad liposomes, with and without polyethylene glycol on the surface of the liposomes, and nitroglycerin as a model vasodilator. The nitroglycerin incorporation efficiency ranged from 25% to 50%. In human sera, liposome formulations with 20mg/mL phospholipid gave rise to complement activation, mainly via the alternative pathway, as reflected by the rises in SC5b-9 and Bb protein complex concentrations. Formulations having a factor of ten lower phospholipid content did not result in measurable complement activation. The weak complement activation induced by Pad-PC-Pad liposomal formulations was confirmed by the results obtained by performing an in vivo study in a porcine model, where hemodynamic parameters were monitored continuously. Our study suggests that, compared to FDA-approved liposomal drugs, Pad-PC-Pad exhibits less or similar risks of CARPA.

Intraocular silicone implant to treat chronic ocular hypotony: an in vivo trial.

PURPOSE: The management of chronic ocular hypotony and complicated proliferative vitreoretinopathy-related retinal detachment represents a challenge. Being non-absorbable and non-biodegradable, a silicone oil implant is expected to restore the volume and the intraocular pressure of the globe, as well as to approximate the detached retina. Further advantages could be a long-term tamponade potential, absence of toxicity, and prevention of silicone oil emulsification or anterior chamber oil-prolapse. The aim of this study was to assess the histological tolerance of the silicone oil implant in a pig model. METHODS: A seamless silicone balloon implant with optional surface modifications was developed. Mini pigs were used as experimental animals, and three variants of silicone implants with different surfaces were tested: uncoated, NCO-sP(EO-stat-PO) coated, and heparin-NCO-sP(EO-stat-PO) coated silicone implants. An extracapsular lens extraction was achieved via a standard phacoemulsification followed by a standard three-port vitrectomy. The implant was then placed in the posterior segment and filled with 5000 centistoke silicone oil. One month later, the pigs were euthanized, the eyes were enucleated, and histological specimens were prepared for microscopy. RESULTS: The analysis of the histology revealed that adverse histological changes in conjunctiva, cornea, iris, and ciliary body could be excluded in all eyes operated on regardless of which variant of implant had been employed. The retina as the implant-contacting ocular tissue showed overall good tolerance, although some inflammatory reaction and fibrous proliferation was evident in some cases. CONCLUSIONS: The silicone oil implant is a promising candidate and has the potential to fulfill clinical requirements to act as a long-term intraocular tamponade agent. The heparin-NCO-sP(EO-stat-PO) coating approach could lead to a novel bioactive surface for intraocular devices with excellent properties to hinder cell adhesion and protein adsorption, although further studies will be necessary to evaluate long-term biocompatibility and long-term resistance to biological attacks.

Cargo self-assembly rescues affinity of cell-penetrating peptides to lipid membranes.

Although cationic cell-penetrating peptides (CPPs) are able to bind to cell membranes, thus promoting cell internalization by active pathways, attachment of cargo molecules to CPPs invariably reduces their cellular uptake. We show here that CPP binding to lipid bilayers, a simple model of the cell membrane, can be recovered by designing cargo molecules that self-assemble into spherical micelles and increase the local interfacial density of CPP on the surface of the cargo. Experiments performed on model giant unilamellar vesicles under a confocal laser scanning microscope show that a family of thermally responsive elastin-like polypeptides that exhibit temperature-triggered micellization can promote temperature triggered attachment of the micelles to membranes, thus rescuing by self-assembly the cargo-induced loss of the CPP affinity to bio-membranes.

Intraocular silicone implant to treat chronic ocular hypotony-preliminary feasibility data.

PURPOSE: Ocular hypotony secondary to proliferative vitreoretinopathy-related retinal detachment, trauma or inflammation is difficult to treat. Besides endotamponades such as silicone oil, vitreous implants such as iris diaphragms or balloons have been developed to stabilize the eye and to prevent phthisis of the globe. Vitreous implants tested thus far exhibit a seam at the attachment site of the hemispheres, or micropores. This manuscript reports the development of a seamless silicone balloon implant without micropores, which can be filled with silicone oil and surface-modified to improve its biocompatibility. Developed for intraocular placement in the management of chronic hypotony and phthisis prevention, it may also be suitable for tamponading retinal detachments. METHODS: Silicone was used as the basic structure for the fabrication of a seamless balloon-shaped intraocular implant, which was coated by employing a six-arm star-shaped (sP) macromer of a copolymer of 80 % ethylene oxide (EO) and 20 % propylene oxide (PO) with conjugated functional terminal isocyanate groups, NCO-sP(EO-stat-PO), with and without heparin. Three variants of implants, which differ in their surfaces, were manufactured: uncoated silicone, NCO-sP (EO-stat-PO) coated silicone and heparin-NCO-sP (EO-stat-PO) coated silicone implants. To exert a tamponade effect, the implant was filled with silicone oil and its properties were studied. RESULTS: Seamless thin balloon implants made of silicone, which are considered biocompatible and intrinsically resistant to biological attacks in vivo, could be fabricated in different sizes. The silicone oil-filled implant can mimic the mechanism of buoyant force and high surface tension of silicone oil, which is the only long-term vitreous substitute currently available. The silicone oil-filled implant can also mimic the natural vitreous body by occupying the entire posterior segment. CONCLUSIONS: The intraocular silicone implant as an alternative long-term treatment of chronic ocular hypotony might offer a new option for clinical ophthalmological practice. In vivo studies need to be performed to collect more data on the implant's long-term mechanical and optical properties, as well as long-term biocompatibility.

Surprising lack of liposome-induced complement activation by artificial 1,3-diamidophospholipids in vitro.

Cardio-vascular diseases are the main cause of death, emphasizing the need to improve patient treatment and survival. One therapeutic approach is a liposome-based drug carrier system specifically targeting constricted arteries. The recently discovered mechano-sensitive liposomes use hemodynamic shear-stress differences between healthy and constricted blood vessels as trigger for drug release. Liposomes are promising delivery containers but are being recognized as foreign by the immune system. Complement activation as essential factor of the recognition leads to adverse effects. Here, we tested complement activation by liposomes formulated from the artificial phospholipid Pad-PC-Pad in vitro. Surprisingly no complement activation was detected in human sera and porcine plasma. In in vivo experiments with three pigs, neither anaphylactic reactions nor other significant hemodynamic changes were observed even at comparably high liposome doses. The pilot study holds promise for an absence of complement-mediated adverse effects of Pad-PC-Pad liposomes in human. FROM THE CLINICAL EDITOR: A lot of research has been done on new treatment for cardiovascular diseases. Liposome-based carrier systems have also shown promises. In this article, the authors studied the potential risks of complement activation by liposomes in in-vivo experiments. The absence of complement activation by Pad-PC-Pad liposomes may indicate its use in humans.

Bilayer properties of 1,3-diamidophospholipids.

A series of 1,3-diamido phosphocholines was synthesized, and their potential to form stable bilayers was investigated. Large and giant unilamellar vesicles produced from these new lipids form a wide variety of faceted liposomes. Factors such as cooling rates and the careful choice of the liposome preparation method influence the formation of facets. Interdigitation was hypothesized as a main factor for the stabilization of facets and effectively monitored by small-angle X-ray scattering measurements.

Thrombophilia in patients with retinal vein occlusion: a retrospective analysis.

BACKGROUND: To assess the prevalence of thrombophilia in patients with central (CRVO) and branch retinal vein occlusion (BRVO). METHODS: In 139 patients with CRVO (n = 88) and BRVO (n = 51) and in 40 healthy controls factor VIII, fibrinogen, antithrombin III, protein C, protein S, activated protein C resistance, anticardiolipin antibodies (ACA), homocysteine, factor V Leiden, prothrombin G20210A and methylene tetrahydrofolate reductase (MTHFR) C677T mutation were assessed retrospectively. RESULTS: Elevated factor VIII activity and the homozygous MTHFR C677T mutation were significantly more often found in CRVO and BRVO cases compared to controls. Age-, gender- and C-reactive protein-adjusted logistic regression analysis did not show a significant additive effect of elevated factor VIII activity on the risk of developing CRVO/BRVO. Elevated fibrinogen levels and ACA were significantly more often found in CRVO than amongst controls. No significant differences were found concerning the remaining variables. CONCLUSIONS: We suggest elevated fibrinogen levels, ACA and the homozygous MTHFR C677T mutation as potential risk factors for CRVO/BRVO.

Human complement factor H and factor H-like protein 1 are expressed in human retinal pigment epithelial cells.

BACKGROUND: A common haplotype in the gene for the regulator of the alternative pathway of complement activation factor H has been linked to individual predisposition to age- related macular degeneration (AMD). METHODS: In this study, retinal pigment epithelial (RPE) cells, i.e. immortalized ARPE-19 as well as primary human RPE cells, were investigated for expression of factor H and FHL-1 by immunohistochemistry and in situ hybridization analysis. RESULTS: Factor H and the alternative spliced product FHL-1 are expressed in RPE cells, i.e. in immortalized ARPE-19 and primary human RPE cells. Factor H and FHL-1 expression was induced in a dose-dependent manner in ARPE-19 cells upon treatment with the inflammatory marker interleukin-6 (IL-6). In situ hybridization experiments confirmed an elevated expression rate of the factor H gene in IL-6-treated ARPE-19 cells. AMD is characterized by complement-associated inflammatory processes in the retina. Thus, local synthesis of complement regulators affects the protection of retinal cells and may be involved in the pathogenesis at the RPE-choroid interface.

Gel-assisted formation of giant unilamellar vesicles.

Giant unilamellar vesicles or GUVs are systems of choice as biomimetic models of cellular membranes. Although a variety of procedures exist for making single walled vesicles of tens of microns in size, the range of lipid compositions that can be used to grow GUVs by the conventional methods is quite limited, and many of the available methods involve energy input that can damage the lipids or other molecules present in the growing solution for embedment in the membrane or in the vesicle interior. Here, we show that a wide variety of lipids or lipid mixtures can grow into GUVs by swelling lipid precursor films on top of a dried polyvinyl alcohol gel surface in a swelling buffer that can contain diverse biorelevant molecules. Moreover, we show that the encapsulation potential of this method can be enhanced by combining polyvinyl alcohol-mediated growth with inverse-phase methods, which allow (bio)molecule complexation with the lipids.

Effects of coagulation on the autofluorescence pattern of ARPE-19 cells: an in vitro study.

OBJECTIVE: Changes in fundus autofluorescence (AF) are observed in various retinal disorders. Lipofuscin accumulation within the retinal pigment epithelium (RPE) is a source of fundus AF (FAF); however, the causes of short-term increases in FAF observed in inflammatory conditions or after laser treatment are unknown. Here, we describe an RPE cell culture model that is useful for investigations of FAF. METHODS: ARPE-19 cells were cultured in 2-well chamber slides. Cells were exposed to isolated rabbit photoreceptor outer segments (POS) to mimic in vivo phagocytic activity. The AF of RPE cells exposed to POS was measured before and after focal coagulation of the cultures. AF was measured over a period of 4 weeks. Cell lysates were examined by two-dimensional (2D) gel electrophoresis and mass spectrometry analysis. RESULTS: The exposure of ARPE cells to POS did not lead to increased AF; however, after coagulation, cells exposed to POS showed a statistically significant increase in AF (p < 0.05). 2D electrophoresis of the cell lysates revealed changes in 3 proteins. One of these proteins, identified by mass spectrometry as ezrin-radixin-moesin-binding phosphoprotein 50, was reduced in the coagulated cell population. CONCLUSIONS: We have established an in vitro model of RPE cells in culture that can be used to evaluate the development of AF and changes in cellular proteins that accompany laser photocoagulation.

Surface-modified silicone foils for intraocular implantation.

BACKGROUND: Implantation of silicone materials like iris diaphragms into the eye can be complicated by cell migration and attachment. We studied polydimethylsiloxane (PDMS) foils coated with isocyanate terminated, star-shaped poly(ethylene glycol-stat-propylene glycol) (NCO-sP(EO-stat-PO)) equipped with heparin towards the inhibition of cell attachment without influencing cell viability. METHODS: Mouse fibroblasts L929 were cultured and seeded onto sterilized pieces of either uncoated NCO-sP(EO-stat-PO) or heparin-NCO-sP(EO-stat-PO) loaded foils. Polyvinylchloride (PVC) foils served as the positive control and biomembranes as the negative control. The cultured cells were examined after 24 h for cell viability and adhesion by fluorescence microscopy; morphological cell changes were documented after hemalaun staining. Cell density was measured and quantification of cell proliferation was assessed by a BrdU test; quantification of cell activity was analyzed by a WST-1 test. RESULTS: The fibroblasts' cell viability was excellent on all tested foils except the toxic PVC foil. NCO-sP(EO-stat-PO) coating provided significantly reduced cell activity. On heparin-loaded coatings, cells were viable and less dense but showed almost the same cell proliferation and cell activity as on the negative control. NCO-sP(EO-stat-PO) coated, heparin loaded foils proved high biocompatibility and reduced cell adhesion. CONCLUSIONS: Both NCO-sP(EO-stat-PO)-coated foils with and without heparin seemed to be a viable implantation material for less cell migration, attachment, and reduced implant complications. Conclusive we give a recommendation for further studies on the intraocular implantation in particular for the NCO-sP(EO-stat-PO)-coated foils.

Early hyperbaric oxygen treatment for nonarteritic central retinal artery obstruction.

PURPOSE: To compare hyperbaric oxygen treatment combined with hemodilution with hemodilution only in central retinal artery obstruction. DESIGN: Retrospective, nonrandomized case series. METHODS: We reviewed records of all our patients diagnosed with central retinal artery obstruction between 1997 and 2010. In these patients, hyperbaric oxygen and hemodilution therapy had been administered routinely (oxygen group). Where hyperbaric oxygenation could not be performed, patients were underwent hemodilution only (control group). Patients with presenting visual acuity (VA) of up to 20/200 within 12 hours of onset were included in our analysis. Exclusion criteria included cilioretinal vessels or arteritic occlusion. RESULTS: The oxygen group comprised 51 patients, and the control group comprised 29 patients. Mean baseline VA was counting fingers (oxygen group) and 20/1000 (control group; P = .1). Most other potential confounders, including duration of symptoms, also did not differ significantly at baseline. In the oxygen group, mean VA improvement was 3 lines (P < .0001). This was sustained over a follow-up of 3 months (P = .01). In the control group, mean improvement was 1 line (P = .23 at discharge, P = .17 at follow-up). Differences between both groups were not significant (P = .07 at discharge, P = .26 at follow-up). The number of patients gaining 3 lines or more was 38.0% versus 17.9% at discharge (P = .06) and 35.7% versus 30.8% at follow-up (P = .76). CONCLUSIONS: We saw significant VA improvement after the combined treatment, but not when using hemodilution only. Confirming superiority of the combination treatment requires a randomized, prospective trial. A high number of nonresponders highlights the need to improve our understanding and treatment of hypoxia-related metabolic insults after central retinal artery obstruction.

Improving the quality of multifocal visual evoked potential results by calculating multiple virtual channels.

PURPOSE: To introduce a method for improvement of multifocal VEP (mfVEP) recordings by prediction of waveforms at multiple positions on the surface of the skull. METHODS: Fifteen healthy participants (mean age 24 ± 3.8 years) underwent mfVEP recordings from 3 surface positions. Two methods of a best-of-mfVEP approach were used and compared. In the first, a standard procedure, further data from 3 calculated channels were used. In the second approach, mfVEPs were obtained by using data derived from 40 virtual electrode positions on the basis of predictions from dipole source calculations. RESULTS: The mean signal-to-noise ratios (SNRs) of the best-of-mfVEPs of both methods were compared. The SNR was significantly higher for mfVEP data using additional virtual recordings revealed by dipole source determination (2.87 vs. 3.36; P < 0.035). CONCLUSION: We conclude that multichannel prediction of mfVEP responses based on dipole source calculation significantly improves the quality of the examination results compared with the currently prevalent standard method.

Detection of heat shock protein 70 in choroidal neovascular membranes secondary to age related macular degeneration.

BACKGROUND: Heat shock proteins are acute phase proteins that are upregulated in inflammation or following thermal stress. We analyzed the presence of the heat shock protein 70 (Hsp 70) in choroidal neovascular (CNV) membranes secondary to AMD after treatment with verteporphin photodynamic therapy (PDT) or transpupillary thermo therapy (TTT) to determine whether treatment correlated with the presence of Hsp70. RESULTS: CNV membranes were removed by pars plana vitrectomy (ppV) and subretinal extraction. The membranes were analysed by light microscopy and the presence of Hsp 70 was examined using histochemistry. HeLa Cells served as controls.Of the 14 membranes analysed 11 were Hsp70 positive and 3 negative. In the no pre-treatment group of 8 membranes 6 were Hsp70 positive and 2 negative; in the PTD group all 4 membranes were positive and in the TTT group 1 membrane was positive and 1 membrane was negative for Hsp70. CONCLUSION: Hsp70 is present in the most CNV membranes secondary to AMD. Pre-treatment of the membrane with PTD or TTT does not appear to influence the expression of Hsp70.

CFH, C3 and ARMS2 are significant risk loci for susceptibility but not for disease progression of geographic atrophy due to AMD.

BACKGROUND: Age-related macular degeneration (AMD) is a prevalent cause of blindness in Western societies. Variants in the genes encoding complement factor H (CFH), complement component 3 (C3) and age-related maculopathy susceptibility 2 (ARMS2) have repeatedly been shown to confer significant risks for AMD; however, their role in disease progression and thus their potential relevance for interventional therapeutic approaches remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here, we analyzed association between variants in CFH, C3 and ARMS2 and disease progression of geographic atrophy (GA) due to AMD. A quantitative phenotype of disease progression was computed based on longitudinal observations by fundus autofluorescence imaging. In a subset of 99 cases with pure bilateral GA, variants in CFH (Y402H), C3 (R102G), and ARMS2 (A69S) are associated with disease (P = 1.6x10(-9), 3.2x10(-3), and P = 2.6x10(-12), respectively) when compared to 612 unrelated healthy control individuals. In cases, median progression rate of GA over a mean follow-up period of 3.0 years was 1.61 mm(2)/year with high concordance between fellow eyes. No association between the progression rate and any of the genetic risk variants at the three loci was observed (P>0.13). CONCLUSIONS/SIGNIFICANCE: This study confirms that variants at CFH, C3, and ARMS2 confer significant risks for GA due to AMD. In contrast, our data indicate no association of these variants with disease progression which may have important implications for future treatment strategies. Other, as yet unknown susceptibilities may influence disease progression.

Effects of fibrinogen and alpha2-macroglobulin and their apheretic elimination on general blood rheology and rheological characteristics of red blood cell aggregates.

Methods of therapeutic apheresis, such as plasma exchange or rheopheresis eliminate moderately aggregating macromolecules like fibrinogen, as well as strongly aggregating substances like alpha2-macroglobulin from blood. In order to examine the specific effect of eliminating alpha2-macroglobulin as a highly aggregating macromolecule, this study aimed to analyze the different rheological properties of: (i) moderately aggregating red blood cells (RBCs; inducible by fibrinogen); and (ii) strongly aggregating RBCs (inducible by alpha2-macroglobulin). In vitro, RBC aggregate geometry was determined in the presence of strong and moderate aggregation inducing macromolecules. In vivo, flow behavior of RBC aggregates was analyzed by intravital microscopy. Using network scanning, the number of perfused and non-perfused microvessels was determined. In vitro, the higher adhesive forces of strongly aggregating RBCs led to both a higher packing density of single RBCs within aggregates, expressed as a significantly reduced thickness of individual RBCs, and greater deformation, expressed as a significantly diminished offset between RBCs and an increased curvature of RBCs at the ends of the aggregates. In vivo rheoscopy showed that only high aggregating RBCs persisted in the precapillary bed and led to the absence of RBCs in up to 40% of nutritive capillaries. These novel findings are of importance regarding recent developments in clinical hemorheology, specifically the clinical use of hemapheretic therapies for diseases in which impaired microcirculation plays a role in either their development or progression, such as age-related macular degeneration and complications of diabetes mellitus. Our data support that procedures reducing the concentration of alpha2-macroglobulin in blood by extracorporeal elimination might provide a more efficient improvement of overall blood fluidity in microcirculatory beds.

Effects of bevacizumab (Avastin) on retinal cells in organotypic culture.

PURPOSE: Repetitive intravitreal injections of bevacizumab are a successful treatment option for exudative age-related macular degeneration (AMD). The aim of this study was to evaluate the toxicity of bevacizumab in the adult mammalian neurosensory retina in culture. METHODS: Adult porcine neurosensory retinas were cultured adjoined to the retinal pigment epithelium-choroid layer (retina-RPE-choroid complex) in static culture for 3 days, whereas neural retinas alone were cultured in a perfusion chamber for 3 days. Bevacizumab was added to the culture and perfusion medium at three concentrations (0.25 mg/mL [n = 6], 0.5 mg/mL [n = 6], and 1.25 mg/mL [n = 6]). Retina-RPE-choroid complex and neural retinas alone cultured without bevacizumab were used as controls. After 3 days in culture, the neural retinas alone and the retina-RPE-choroid complexes were analyzed histologically and immunohistochemically for the expression of glial fibrillary acidic protein (GFAP), vimentin, glutamine synthetase, rhodopsin, smooth muscle actin (SMA), and apoptosis. RESULTS: No toxic effects on ganglion or photoreceptor cells were observed at any concentration of bevacizumab. The expression of GFAP and vimentin was slightly increased in Müller cells, whereas glutamine synthetase and rhodopsin were unaffected by bevacizumab. However, significantly enhanced SMA expression in retina blood vessels was observed in retinas cultured in the presence of bevacizumab. CONCLUSIONS: Bevacizumab was well tolerated by ganglion and photoreceptor cells even at concentrations fivefold higher than those used clinically. The increased expression of SMA is an indication of the loss of functional VEGF modulating smooth muscle cells in mature vessels.