Analysis of the neuronal marker protein gene product 9.5 in internal limiting membranes after indocyanine-green assisted peeling.

BACKGROUND: Indocyanine green-assisted internal limiting membrane (ILM) peeling was suspected to disrupt the innermost layer of the neural retina. We examined whether surgically excised specimens contain remnants of neuronal tissue. METHODS: Ten patients with macular hole underwent pars plana vitrectomy and indocyanine green-assisted ILM peeling. A total of 0.1 mL of a 0.5% indocyanine green solution was applied for 15 seconds. The ILM specimens were prepared for immunohistochemistry, using a polyclonal antibody against protein gene product 9.5. Protein gene product 9.5 is a pan-neuronal marker labeling human neuronal cells. Appropriate controls to show selectivity of the antibody were performed on neuronal tissue of donor eyes. One ILM was prepared for electron microscopy. RESULTS: A selective expression of protein gene product 9.5 was found in neuronal fibers of the retina and optic nerve of donor eyes. Only 1 of the 10 surgical ILM specimens showed a minimal focal positivity for protein gene product 9.5. No neuronal tissue was detected on the ILM by electron microscopy. CONCLUSION: Focal expression of protein gene product 9.5 in only 1 of 10 surgical ILM specimens argues against a general indocyanine green-related disruption of the innermost retinal layers. However, higher concentrations of the dye, longer incubation times or different solvents than used in this study may lead to different results.

Intensified monitoring of circadian blood pressure and heart rate before and after intravitreous injection of bevacizumab: preliminary findings of a pilot study.

BACKGROUND: A dose-dependent increase in arterial blood pressure (BP) was seen during bevacizumab treatment given intravenously for metastatic carcinoma. Because low systemic levels can also be expected after the intravitreal administration of bevacizumab, we looked for possible haemodynamic reactions of patients at higher risk of developing cardiovascular events after bevacizumab injection. METHODS: Ambulatory BP was monitored in 14 hypertensive patients receiving 1.25 mg intraocular bevacizumab for either choroidal neovascularization (CNV) or retinal proliferation associated with central retinal vein occlusion (CRVO). Circadian measurement was carried out twice, first at least 24 h prior to injection and second 72 h afterwards. Baseline evaluation before injection was compared with values taken in a matched control group. Taking a small random sample of two patients, serum concentration of bevacizumab and VEGF-A was measured at several time points. RESULTS: High incidence of pathologic BP values was found in the pre-injection measurement, even under anti-hypertensive treatment of the patients with CNV or CRVO. No general increase in BP was seen after the intravitreal injection (P = 0.01), although significantly reduced nocturnal dipping occurred as compared to before the injection (P = 0.006). Individual patients showed a rise in BP load subsequent to injection. A decline in serum VEGF-A was found to correspond to measureable levels of serum bevacizumab (up to 90 ng/ml). CONCLUSIONS: Before the intravitreal injection, BP values were increased in the majority of the patients. The elevated BP load might be related to probable pre-injection stressors. There seems to be no general rise in mean BP, heart rate and pulse pressure after intravitreal bevacizumab, although a decrease in serum VEGF-A can occur in individual patients. The reduced nocturnal dipping could be caused by pharmacodynamic effects on the vasal tone; this preliminary but striking finding warrants further investigation.

Safety parameters for indocyanine green in vitreoretinal surgery.

Since the early nineties removal of the internal limiting membrane (ILM) has been shown to be an effective and safe treatment option for conditions that involve the vitreoretinal interface. Peeling of the barely visible ILM, however, represents a challenge and complete removal is difficult and not always obvious. Damage at the vitreoretinal interface or unsatisfactory peeling may therefore be the result of the genuine procedure. Introduction of indocyanine green (ICG) for ILM staining led to better visibility of the ILM and greatly facilitated this surgical maneuver making ILM peeling more controllable, easier and faster. Consequently, enthusiastic acceptance resulted in an uncritical use not supported by preclinical safety data. Soon thereafter some clinical reports raised concerns about potential cytotoxic effects related to the intravitreal use of ICG. The following chapter summarizes the results of in vitro, ex vivo, in vivo and clinical studies related to the use of ICG in vitreoretinal surgery. Critical appraisal of the methodical procedures and results leads to the nonnegligible fact that ICG has a cytotoxic effect enhanced by photoactivation. The results of several studies as well as our experimental workup, however, showed that ICG toxicity to the retinal pigment epithelium is dependent on the dye concentration, the osmolarity of the solvent solutions, as well as on the lengths of dye exposure time and of the vitrectomy endolight illumination time. With respect to the safety margins and profile, ICG is therefore a useful surgical tool that is still widely applied, but that may be replaced by more inert and as efficient vital dyes.

Penetration of bevacizumab through the retina after intravitreal injection in the monkey.

PURPOSE: The penetration of intravitreally injected bevacizumab in its commercial formulation (Avastin; Roche, Grenzach, Germany) through the retina was studied, to determine whether a full-length antibody would be able to penetrate the retina as easily as an antibody fragment. METHODS: Six cynomolgus monkeys (Macaca fascicularis) were used in this study. Two compositions of intravitreal injection into the right eyes were performed: one with commercial Avastin (group 1, four animals) and the other one with commercial Avastin labeled with 125I (group 2, one animal). The animals in group 1 were killed 1, 4, 7, or 14 days after the injection for subsequent histologic analysis of the eyes by immunohistochemistry, and the animal in group 2 was killed 7 days after injection for autoradiography and electron microscopy. Funduscopy was performed before the injection and at several time points thereafter. Moreover, blood samples were collected at different time points from the group-2 animal. The sixth animal remained untreated and served as the control. RESULTS: No pathologic changes were obvious in the funduscopic images within the time of the experiment. Bevacizumab immunoreactivity was found in the choroid and the inner layers of the retina as early as 1 day after the injection and spread to the outer layers and the choroid within the following days, in particular to photoreceptors and blood vessels. Avastin labeled with 125I showed radioactivity in blood serum 1 day after the intravitreal injection and remained relatively stable until day 7. CONCLUSIONS: The results clearly show that the bevacizumab molecule can penetrate the retina and is also transported into the retinal pigment epithelium, the choroid and, in particular, into photoreceptor outer segments after intravitreal injection of Avastin. Active transport mechanisms seem to be involved.

Angiopoietin modulation of vascular endothelial growth factor: Effects on retinal endothelial cell permeability.

PURPOSE: Vascular permeability is important at many sites, but particularly so in diabetic retinopathy where macular oedema is the major cause of blindness. Angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) are important factors involved in neovascularization and vascular leakage, but there is little data on their interaction to promote increased vascular permeability. METHODS: Porcine retinal endothelial cells (PREC) were seeded into permeable inserts and cultured in 24-well plates that permit measurement of permeability using fluorescent dextrans. Cell purity was assessed immunohistochemically. At confluency, PREC were treated with increasing concentrations of VEGF (20-100ng/ml) and Ang-2 (15-75ng/ml). The effect on tight junctions was assessed by visualization with an anti-ZO-1 antibody. RESULTS: Immunohistochemistry showed high purity of isolated PREC. Permeability of untreated PREC monolayers was low. The increase in permeability in Ang-2 treated cells (25-30% compared with non-treated cells) was less than that for cells treated with VEGF only (20-100% compared with untreated cells). Highest permeability was seen with a combination of Ang-2 and VEGF (100-400% compared with untreated cells). Permeability increased with time after growth factor application. Preliminary ZO-1 immunohistochemistry appeared to demonstrate the presence of tight junctions between untreated PREC, and loss of tight junctions after treatment with VEGF and Ang-2. CONCLUSIONS: VEGF alone is twice as potent in interrupting tight junctions in an endothelial cell monolayer as Ang-2. However, both growth factors acting together increase permeability three times as much as VEGF alone. Treatments designed to reduce vascular permeability in diabetic macular oedema should consider that crosstalk between growth factors including VEGF and the Ang-2/Tie-2 system can multiply their effects.

Ultrastructural findings in the primate eye after intravitreal injection of bevacizumab.

PURPOSE: To examine the ultrastructural effect of intravitreal bevacizumab on primate eyes with particular focus set on the choriocapillaris and to examine the influence of vascular endothelial growth factor (VEGF) inhibition on endothelial cell fenestration. DESIGN: Animal study. METHODS: Four Cynomolgus monkeys received an intravitreal injection of 1.25 mg bevacizumab. The eyes were enucleated and prepared for light and electron microscopy on days one, four, seven, and 14. Control eyes remained untreated. Choriocapillaris endothelial cell fenestrations were quantified. RESULTS: Choriocapillaris endothelial cell fenestrations were significantly reduced after intravitreal injection of bevacizumab. Fenestration was lowest on day four (15.9 +/- 6.7 per 25 microm) and increased again from days seven to 14, but was still significantly lower than in the control (66.2 +/- 9.5 per 25 microm). Densely packed thrombocytes and leukocytes regionally occluded the choriocapillaris lumen of treated eyes. On day one an increased number of leukocytes filled in the choriocapillaris lumen. Photoreceptors were damaged in two of 40 light microscopic sections. On days one to seven, choroidal melanocytes contained giant melanosomes. None of these described features was found in controls. CONCLUSIONS: Intravitreal bevacizumab causes ultrastructural changes in the choriocapillaris of primate eyes. A significant reduction of choriocapillaris endothelial cell fenestrations is seen as early as 24 hours after injection and their number increases again after two weeks. These findings may play a role in the early clinical effect of intravitreal bevacizumab for macular edema. Because an increased risk of circulation disturbances in the choriocapillaris cannot be excluded, patients should be carefully monitored.

Antipermeability and antiproliferative effects of standard and frozen bevacizumab on choroidal endothelial cells.

BACKGROUND: Bevacizumab is an antiangiogenic compound developed to target tumour vessels. Its off-label use in ophthalmology requires in vitro testing on ocular cells. AIM: To quantify the antipermeability and antiproliferative effects of bevacizumab on cultured choroidal endothelial cells (CECs). It was examined whether deep-freezing of bevacizumab attenuates its antiangiogenic activity. METHODS: Porcine CECs were cultured in permeable insert systems. Permeability of the cell monolayers was quantified by a fluorescent isothiocyanate-dextran assay after treatment with vascular endothelial growth factor (VEGF; 20-100 ng/ml) alone and in combination with bevacizumab (0.1-1 mg/ml). Proliferation of the CECs was tested using a "wound scratch" assay. The experiments were repeated with bevacizumab after freezing at -20 degrees C for 5 days. RESULTS: Bevacizumab significantly reduced VEGF-induced permeability in a dose-dependant manner. A molar ratio of 2.6:1 of bevacizumab to VEGF was required for complete blocking of VEGF-induced rise in permeability. CEC proliferation was significantly blocked by bevacizumab (0.5 mg/ml). Thawed bevacizumab after deep freezing showed a moderate, but not statistically significant loss in activity. CONCLUSION: Bevacizumab significantly reduces VEGF-induced permeability and proliferation of CECs. Freezing and thawing of bevacizumab will affect its biological activity.

The role of PDGF receptor inhibitors and PI3-kinase signaling in the pathogenesis of corneal neovascularization.

PURPOSE: Corneal neovascularization remains an unsolved therapeutic problem. Platelet-derived growth factor (PDGF) is directly linked to vessel formation and stabilization. This study was undertaken to elucidate the mechanisms by which PDGF exerts its effects on corneal angiogenesis. METHODS: Corneal neovascularization was induced in C57 mice by removal of the limbal epithelium. When mature vessels appeared after 7 days, mice were treated with the PDGF receptor-beta inhibitor AG 1296 or the phosphatidylinositol 3-kinase (PI3-K)-inhibitors wortmannin and LY294002, respectively, using an intraperitoneally implanted miniosmotic pump. At day 14 after scraping, corneas of treated and untreated (control) mice were dissected and immunostained with FITC-CD31 antibody for endothelial cells and with Cy3-SMA (smooth muscle actin) for pericytes. VEGF (vascular endothelial growth factor), ang1/2 (angiopoietin 1 and 2), and PDGF mRNA levels of treated and untreated corneas were determined by real-time RT-PCR. RESULTS: Mice treated with the PDGF inhibitor AG 1296 showed an inhibition of corneal neovascularization of 21.1% and a reduction of pericytes of 52% in the newly formed vessels compared with untreated animals. VEGF, ang1, ang2, and PDGF mRNA expression was reduced in the corneas of AG 1296-treated mice compared with the respective control. Treatment with the PI3-K inhibitors wortmannin and LY29002 had similar effects, inducing a decrease in corneal neovascularization and a reduction of VEGF, ang1, ang2, and PDGF mRNA levels. CONCLUSIONS: Inhibition of the PDGF signal pathway results in loss of pericytes and a reduction in vessel density in the neovascularized cornea that correlates with reduced expression of PDGF, ang1/2, and VEGF mRNA. Furthermore, PI3-K was shown to be involved in the regulation of VEGF, ang1, and PDGF, as the PI3-K inhibitors wortmannin or LY294002 had similar effects. Because PDGF is a known stimulus for PI3-K activation, it can be postulated that the observed decrease in VEGF, ang1/2, and PDGF mRNA levels on administration of the PDGF inhibitor is caused by the decreased activation of the PI3-K signaling cascade.

Intracameral bevacizumab for iris rubeosis.

PURPOSE: To determine whether intracameral bevacizumab decreases vascular leakage from iris rubeosis in patients with neovascular glaucoma. DESIGN: Interventional case series. METHODS: The study included six eyes of three patients with secondary neovascular glaucoma due to proliferative diabetic retinopathy (n = 2) or ischemic central retinal vein occlusion (n = 1). All patients received an intracameral injection of 1.0 mg bevacizumab. Morphologic changes and vascular leakage were investigated prospectively by iris fluorescein angiography. RESULTS: Decrease in leakage was detected as early as one day after injection. No inflammation was observed. No relapse was seen within the follow-up of four weeks. CONCLUSION: Intraocular injection of bevacizumab may provide an additional strategy for the treatment of iris rubeosis in neovascular glaucoma.

Melanin protects choroidal blood vessels against light toxicity.

Low ocular pigmentation and high long-term exposure to bright light are believed to increase the risk of developing age-related macular degeneration (ARMD). To investigate the role of pigmentation during bright light exposure, cell damage in retinae and choroids of pigmented and non-pigmented rats were compared. Pigmented Long Evans (LE) rats and non-pigmented (albino) Wistar rats were exposed to high intensity visible light from a cold light source with 140,000 lux for 30 min. Control animals of both strains were not irradiated. The animals had their pupils dilated to prevent light absorbance by iris pigmentation. 22 h after irradiation, the rats were sacrificed and their eyes enucleated. Posterior segments, containing retina and choroid, were prepared for light and electron microscopy. Twenty different sections of specified and equal areas were examined in every eye. In albino rats severe retinal damage was observed after light exposure, rod outer segments (ROS) were shortened and the thickness of the outer nuclear layer (ONL) was significantly diminished. Choriocapillaris blood vessels were obstructed. In wide areas the retinal pigment epithelium (RPE) was absent in albino rats after irradiation. In contrast, LE rats presented much less cell damage in the RPE and retina after bright light exposure, although intra-individual differences were observed. The thickness of the ONL was almost unchanged compared to controls. ROS were shortened in LE rats, but the effect was considerably less than that seen in the albinos. Only minimal changes were found in choroidal blood vessels of pigmented rats. The RPE showed certain toxic damage, but cells were not destroyed as in the non-pigmented animals. The number of melanin granules in the RPE of LE rats was reduced after irradiation. Ocular melanin protects the retina and choroid of pigmented eyes against light-induced cell toxicity. Physical protection of iris melanin, as possible in eyes with non-dilated pupils, does not seem to play a major role in our setup. Biochemical mechanisms, like reducing oxidative intracellular stress, are more likely to be responsible for melanin-related light protection in eyes with dilated lens aperture.

Investigation of laser-induced choroidal neovascularization in the rat.

PURPOSE: Choroidal neovascularization plays an important role in pathogenesis of age-related macular degeneration. Induction of neovascularization by laser photocoagulation in the rat fundus is an established animal model in which the effects of new therapeutic approaches are assessed. The purpose of this study was to compare different detection methods of laser-induced neovascularization in the rat. METHODS: Laser spots were applied to the fundus of Long-Evans rats. Ten days after, four different methods were used to detect laser-induced neovascularization: (1) high-resolution angiography with fluorescein isothiocyanate-dextran, (2) immunohistochemical visualization of platelet endothelial cell adhesion molecule (PECAM)-1, (3) visualization of intravascular lumens by peroxidase perfusion in the living rat with subsequent histologic analysis, and (4) histochemical representation of alkaline phosphatase in endothelial cells. RESULTS: At the rim of the laser scars vessel-forming endothelial cells with intravasal dextran and peroxidase were present. Cross-sections demonstrated that these vessels originated from the retina. The center of the scars contained homogenous endothelial cells of choroidal origin, which was confirmed by immunohistochemistry and electron microscopy. In laser-treated eyes without FITC-dextran perfusion, scars showed unspecific fluorescence, making differentiation from specific FITC-dextran-associated fluorescence difficult. CONCLUSIONS: In the rat model of laser-induced neovascularization, newly developed endothelial cells originate from the retina and the choroid. Whereas ring-like surrounding vessels come from the retina, flat endothelial cells in deeper layers are of choroidal origin or may originate from circulating endothelial precursor cells. Dextran angiography has to be regarded critically for visualizing the choriocapillaris and CNV in laser scars. PECAM-1 immunohistochemistry is best for detection and quantification of neovascularization in laser scars.

Bevacizumab as adjuvant for neovascular glaucoma.

PURPOSE: We aimed to evaluate the longterm effects of intraocular bevacizumab (Avastin) injections as adjuvant treatment in patients with neovascular glaucoma. METHODS: Twenty eyes of 18 consecutive patients with secondary neovascular glaucoma caused by proliferative diabetic retinopathy (n = 7), ischaemic central retinal vein occlusion (n = 7), ischaemic ophthalmopathy (n = 2) and retinal ischaemia resulting from persistent detachment (n = 2) were treated with intraocular bevacizumab injections (1.25 mg/0.05 ml) in addition to other treatments. The main outcome measure was the change in degree of iris rubeosis. Secondary outcomes included intraocular pressure (IOP), best corrected visual acuity (BCVA) and numbers of additional interventions or antiglaucoma medications administered after injection. RESULTS: Mean (+/- standard deviation) follow-up was 67.7 +/- 13.8 weeks (range 50-93 weeks). At the last follow-up, complete regression of rubeosis was detectable in five (20%) eyes, incomplete regression in seven (35%), stabilization in six (30%), and an increase in two (10%) eyes. Mean IOP was 26.0 +/- 8.9 mmHg at baseline and significantly decreased to 14.75 +/- 5.3 mmHg at the last follow-up visit (p = 0.000005). Mean baseline BCVA (logMAR [logarithm of the minimum angle of resolution] 1.43 +/- 0.89) was stabilized during the follow-up period (logMAR 1.5 +/- 0.98). Patients received an average of 2.75 injections. Additional treatments were laser photocoagulation in 13 (65%) eyes, cyclodestructive procedure in 14 (70%), cryopexy in six (30%), drainage procedures in two (10%), and vitrectomy in five (25%) eyes. CONCLUSIONS: Bevacizumab may be beneficial as adjuvant treatment in neovascular glaucoma because of its anti-angiogenic properties and its ability to prevent establishment or progression of angular obstruction. The causative disease inducing the angiogenic process requires treatment in all cases. Antiglaucoma treatment is needed in cases of persistent elevated IOP.

Electrophysiological effects of Brilliant Blue G in the model of the isolated perfused vertebrate retina.

BACKGROUND: To facilitate epiretinal or inner limiting membrane peeling dyes like Indocyanine Green (ICG) as well as Trypan Blue (TB) were used so far. However, toxic effects on the retina were described for both dyes. The aim of our study was to investigate the retinal tolerance to the new dye Brilliant Blue G (BBG), which implies a possibly more favorable biocompatibility. METHODS: Bovine retina preparations were perfused with an oxygen preequilibrated standard solution. The electroretinogram (ERG) was recorded using Ag/AgCl-electrodes. After recording stable b-wave amplitudes, Brilliant Blue G (BBG) was applied epiretinally for 10 s, 15 s, 30 s, 60 s and 120 s at a concentration of 0.25 mg/ml, which was recently proposed for vitreoretinal surgery. To disclose the effects of BBG on photoreceptor function two test series at the same concentration were performed to evaluate the impact of BBG on the a-wave amplitude. Aspartate at a concentration of 1 mM was added to the nutrient solution to obtain stable a-wave amplitudes. Thereafter, BBG was epiretinally applied for 30 s or 60 s. The recovery of the ERG-amplitudes was followed up for 115 min. RESULTS: Reductions of the a- and b-wave amplitude were found directly after exposure with BBG in each test series, but the effects on the electroretinogram after application of BBG were rapidly and completely reversible within the recovery time for all exposure times. No differences were found between the ERG-amplitudes before and after dye application at the end of the washout. CONCLUSIONS: BBG seems to be an alternative vital staining dye with a good biocompatibility. Comparing the effects with Indocyanine Green or Trypan Blue in the model of the isolated perfused vertebrate retina BBG exhibits a more favorable safety profile.

The effects of triamcinolone crystals on retinal function in a model of isolated perfused vertebrate retina.

A good clinical experience of intravitreal triamcinolone acetonide (TA) has been reported in several studies, but there are growing indications that epiretinal crystals of TA exhibit retinal toxicity. To investigate the effects of TA on retinal function we used a model of an electrophysiological in vitro technique for testing retinal toxicity. Isolated bovine retinas were perfused with an oxygen saturated nutrient solution. The electroretinogram (ERG) was recorded as a transretinal potential using Ag/AgCl electrodes. After reaching stable ERG-amplitudes TA at the maximum solubility equilibrium (36 microg/ml) was either applied to the nutrient solution for 45 min or TA was administered epiretinally at concentrations (1 mg/ml, 4 mg/ml, 8 mg/ml, 20 mg/ml and 40 mg/ml) above the maximum solubility equilibrium to assure direct contact of the TA crystals with the isolated perfused retinas. After that the retinas were reperfused for 75 min with the standard nutrient solution. The percentage of a- and b-wave reduction directly after the application and at the washout was calculated. To assess the effects of TA at the level of the ganglion cell layer a Viability/Cytotoxicity Kit for mammalian cells was used. No changes of the ERG-amplitudes were detected during the exposure to 36 microg/ml TA for 45 min (b-wave: 9.6 microV+/-2.1 vs. 8 microV+/-2.1 (p=0.135); a-wave: -11 microV+/-2.7 vs. -10.6 microV+/-2.3 (p=0.889)) and at the washout (b-wave: 8 microV+/-2.1 vs. 8.3 microV+/-2.4 (p=0.18); a-wave: -10.6 microV+/-2.3 vs. -12 microV+/-2.6 (p=0.225)). At concentrations higher than 1mg/ml TA induced a decrease of the a- and b-wave in a concentration dependent manner. These changes were reversible for concentrations of TA up to 20mg/ml (b-wave: 9 microV+/-2.4 vs. 6.6 microV+/-2.5 (p=0.08); a-wave: -11.4 microV+/-2.0 vs. -11.2 microV+/-2.2 (p=0.37)), but irreversible at 40 mg/ml even at the end of the washout (b-wave: 9.8 microV+/-1.9 vs. 3 microV+/-1.7 (p=0.009); a-wave: -9.8 microV+/-2.1 vs. -2.6 microV+/-2.1 (p=0.001)). Histological examination of the preparations revealed a dramatic ganglion cell death, in which an application of 20mg/ml and 40 mg/ml TA led to a 60.53% (p=0.013) and 82.35% (p=0.002) ganglion cell death, respectively. The epiretinal application of 4 mg/ml TA and higher resulted in distinct effects on the ERG of the isolated perfused retinas. Ganglion cell death was induced at a concentration of 20mg/ml and higher. TA shows an asymmetric and partly high concentrated distribution after intravitreal application. Therefore, we consider concentrations of 4 mg/ml and higher might be toxic and should be avoided in clinical use.

Systematic evaluation of ICG and trypan blue related effects on ARPE-19 cells in vitro.

The aim of our study was a systematic analysis of the impact of variable parameters on indocyanine green (ICG) and trypan blue (TB) related cytotoxicity on human RPE cells. ARPE-19 cells were incubated with ICG (5.0-0.025mg/ml), with ICG-free solutions of corresponding osmolarities or with TB (1.5-0.0375mg/ml). Incubation lasted 1-20min with or without endolight illumination for 1min or 5min. Cell viability and morphology were examined after 6h, 24h and 72h to detect acute and delayed effects. In the absence of endolight, ICG cytotoxicity depends on osmolarity and exposure time. In the presence of endolight, cytotoxic effects are influenced by dye concentration. TB cytotoxicity depends on dye concentration and exposure time, but not on illumination. All observed cytotoxic effects were mainly acute. Both ICG and TB can be cytotoxic depending on concentration and exposure time. ICG related cytotoxic effects are additionally determined by osmolarity and phototoxicity. However, concentrations (<1mg/ml) and incubation times (<5min) as used in clinical practice would appear to be well tolerated.

Inhibition of lysosomal degradation in retinal pigment epithelium cells induces exocytosis of phagocytic residual material at the basolateral plasma membrane.

PURPOSE: To analyze chloroquine-induced morphological changes in the retinal pigment epithelium (RPE) and Bruch's membrane (BM). METHODS: Retina-choroid complexes of chloroquine-treated Long-Evans rats were analyzed by electron microscopy. RESULTS: Intercellular spaces between the RPE cells and BM were enlarged. Residual material from phagosomes was released into these enlarged spaces. Debris accumulated within BM and encircled choriocapillaris endothelial cells. CONCLUSION: There is a release of undegraded phagocytic material (rod outer segments) into the extracellular space between BM and RPE cells, following inhibition of lysosomal degradation. Electron-dense deposits in BM and choriocapillaris may lead to reduced oxygen and nutrition flow.

Tyrosinase biosynthesis in adult mammalian retinal pigment epithelial cells.

Tyrosinase (EC 1.14.18.1) is the rate limiting enzyme of melanogenesis and it is unclear whether it is synthesized in postnatal retinal pigment epithelium (RPE). Cultured RPE cells from cattle were fed with isolated rod outer segments (ROS). After phagocytosis, RPE cells were tested for tyrosinase presence and activity with three independent methods: (1) ultrastructural DOPA (l-3,4-dihydroxyphenylalanine) histochemistry (2) immunocytochemistry with anti-tyrosinase antibodies (3) measuring tyrosine hydroxylase activity using [(3)H]tyrosine. With all three methods tyrosinase was found in RPE cells after ROS-feeding but was absent without feeding. In contrast to the classical hypothesis, we demonstrated with three independent methods that the expression of tyrosinase and its enzymatic activity are induced in cultured adult RPE by phagocytosis of rod outer segments (ROS) in vitro.