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Purpose: To test the intracameral safety of nepafenac and its efficacy in inhibiting prostaglandin synthesis during phacoemulsification surgery., Methods: The safety evaluation was conducted in normal eyes of rabbits, 0.1ml of 0.3% and 1% nepafenac was injected intracamerally. Extensive studies to detect adverse response ranged from a gross examination of eyes under slit lamp biomicroscope, fluorescein dye test, Schirmer tear test, test for corneal sensitivity, intraocular pressure measurement (IOP), specular microscopy, electroretinography(ERG), and histopathological examination of intraocular tissues. Efficacy of nepafenac was studied by intracameral injection of 0.1%, 0.3% nepafenac, nepafenac 0.3%+1% lignocaine, and 1% lignocaine alone, before phacoemulsification surgery and intraoperative mydriasis along with PGE2(ProstaglandinE2) secretion were recorded., Results: Single 0.1ml of 0.3% or 1% nepafenac did not significantly (p > 0.05) alter physiological parameters and histology of cornea, iris, and retina. Nepafenac 0.3% effectively inhibited PGE2 secretion. No significant (p > 0.05) prevention of miosis was recorded with 0.1% or 0.3% nepafenac. However, a combination of 0.3% nepafenac + 1% lignocaine and 1% lignocaine alone significantly (p < 0.05) arrested miosis during the intraoperative period., Conclusion: An intracameral concentration of up to 1% nepafenac does not adversely affect the rabbit eye. Nepafenac fails to prevent miosis but inhibits prostaglandin release during phacoemulsification surgery.

Purpose: Retinitis pigmentosa (RP) is a group of inherited diseases characterized by the death of rod photoreceptors, followed by the death of cone photoreceptors, progressively leading to partial or complete blindness. Currently no specific treatment is available for RP patients. Sulforaphane (SFN) has been confirmed to be an effective antioxidant in the treatment of many diseases. In this study, we tested the therapeutic effects of SFN against photoreceptor degeneration in Pde6b rd10 mice., Methods: rd10 mice and C57/BL6 wild-type (WT) mice were treated with SFN and saline, respectively, from P6 to P20. Electroretinography (ERG), terminal deoxynucleotidyl transferase dUTP nick end labeling and western blot were tested, respectively, at P21 for the analysis of retinal function, retinal cell apoptosis or death and the protein express of GRP78/BiP (TUNEL) as a marker of endoplasmic reticulum (ER) stress., Results: Compared with the saline group, the SFN-treated group showed significantly higher ERG a-wave and b-wave amplitudes, less photoreceptor death, and the downregulation of GRP78/BiP., Conclusions: Our data showed that SFN ameliorated the retinal degeneration of rd10 mice, which is possibly related to the downregulation of GRP78 expression.

Purpose: Longer-lasting electroretinographic recordings of the isolated murine retina were initially achieved by modification of a phosphate-buffered nutrient solution originally developed for the bovine retina. During experiments with a more sensitive mouse retina, apparent model-specific limitations were addressed and improvements were analyzed for their contribution to an optimized full electroretinogram (ERG)., Material and Methods: Retinas were isolated from dark-adapted mice, transferred to a recording chamber and superfused with different solutions. Scotopic and photopic ERGs were recorded with white flashes every 3 minutes. The phosphate buffer (Sickel-medium) originally used was replaced by a carbonate-based system (Ames-medium), the pH of which was adjusted to 7.7-7.8. Moreover, addition of 0.1 mM BaCl 2 was investigated to reduce b-wave contamination by the slow PIII component typically present in the murine ERG., Results: B-wave amplitudes were increased by the pH-shift (pH 7.4 to pH 7.7) from 22.9 ± 1.9 µV to 37.5 ± 2.5 µV. Improved b-wave responses were also achieved by adding small amounts of Ba 2+ (100 µM), which selectively suppressed slow PIII components, thereby unmasking more of the true b-wave amplitude (100.0% with vs. 22.2 ± 10.7% without Ba 2+ ). Ames medium lacking amino acids and vitamins was unable to maintain retinal signaling, as evident in a reversible decrease of the b-wave to 31.8 ± 3.9% of its amplitude in complete Ames medium., Conclusions: Our findings provide optimized conditions for ex vivo ERGs from the murine retina and suggest that careful application of Ba 2+ supports reliable isolation of b-wave responses in mice. Under our recording conditions, murine retinas show reproducible ERGs for up to six hours.

Purpose: Evaluate toxicity of acai fruit (Euterpe oleracea) dye concentrations in a rabbit model., Methods: Rabbits were injected intravitreously with 10%, 25%, and 35% acai dye concentrations. Control eyes received balanced salt solution (BSS). Electroretinogram (ERG), fundus imaging, fluorescein angiography (FA), optical coherence tomography (OCT), and light and transmission electron microscopy (LM/TEM) were performed., Results: Fundus imaging showed increased vitreous opacity with increased dye concentrations. FA and OCT showed normality with all concentrations. Comparisons between BSS and dye concentrations were analyzed using Kruskal-Wallis and Mood's median test (p < 0.05). At 24 h, ERGs showed reduced amplitudes from baseline in all eyes. Median b-wave amplitudes nonsignificantly decreased and latency increased with 10% and 25%; findings were significant (p < 0.05) for 35%. LM and TEM showed no abnormalities for 10% and 25%. With 35%, TEM showed ganglion cell edema at 24 h that resolved after 7 days. Vacuolization, multilamellar bodies, and nerve bundle damage occurred at 24 h/7 days in the inner nuclear layer. Mitochondrial cristae disruption occurred in the inner photoreceptor segment at 24 h that decreased by 7 days., Conclusion: Ten and twenty-five percent concentrations were safe and may improve identification of the posterior hyaloid and internal limiting membrane during chromovitrectomy in humans.

Purpose: Granulocyte colony-stimulating factor (G-CSF) has potential ocular neuroprotective effects. The aim of this study was to evaluate the retinal toxicity of intravitreal G-CSF in rabbit eye., Methods: Eight New Zealand albino rabbits, weighing between 2 and 3 kg, were selected for this study. The initial concentration of G-CSF (300 µg/0.5 ml) was titrated to obtain different concentrations of 45 μg, 30 μg, 15 μg, and 7.5 μg in 0.1 ml. Each concentration was injected into two rabbit eyes. For each dose, dextrose was injected in one contralateral eye and the other fellow eye remained non-injected. Electroretinographic (ERG) testing was performed before and 4 weeks after injections. The rabbits were euthanized and the eyes were enucleated 4 weeks after injections and examined using light microscopy and immunohistochemistry., Results: One rabbit with the injected dosage of 7.5 µg died at the first post-injection day. No sign of intraocular toxicity was found in clinical examination in other rabbits. A significant decrease in at least one of the a- or b-wave measurements of scotopic or photopic responses was found in 45 µg, 15 µg, and 7.5 µg injected eyes. Eyes with an intravitreal injection dosage of 30 µg G-CSF did not have significant changes compared to the baseline values. Histologic and immunohistochemistric studies were unremarkable for pathologic changes in all injected eyes., Conclusion: While histologic and immunohistochemistric examinations revealed no toxicity in all G-CSF-injected eyes, significant ERG changes were observed in all doses except for the dose of 30 µg/0.1 ml.

Purpose: To investigate changes in retinal ganglion cell (RGC) activity by measuring the positive scotopic threshold response (pSTR) of the electroretinogram (ERG) in axotomized eyes after brimonidine injection., Methods: In 50 adult Sprague-Dawley rats, the left eye was axotomized and injected with phosphate buffered saline (PBS) or brimonidine and the contralateral right eye was left untreated. Scotopic ERGs were recorded simultaneously from both eyes on days 1, 2, 3, 7, and 10 after the intravitreal injection, and the amplitude of the a- and b-waves and the pSTR were measured. Surviving RGCs in the flat-mounted retinas were counted 10 days after axotomy. In addition to brimonidine, K252a (an inhibitor of tyrosine kinase phosphorylation of the Trk receptors), U0126 (a MAPK/ERK kinase inhibitor), and LY294002 (phosphoinositide 3-kinases [PI3Ks]) were also injected intravitreally into the left eye, and ERGs were recorded using the same protocol., Results: The pSTR amplitude increased significantly in the axotomized eyes with brimonidine, to 122.9 ± 5.0%, 161.8 ± 8.3%, and 133.6 ± 8.1% on days 1, 2, and 3 (P < 0.01), respectively, compared to the axotomized eyes treated with PBS (control). The increased pSTR amplitude returned to normal (103.6 ± 6.7%) on day 7, although there were a greater number of surviving RGCs in the treatment groups than in the controls. The intravitreal injection of K252a, U0126, or LY294002 significantly attenuated the increase in pSTR induced by intravitreal brimonidine (P < 0.01)., Conclusion: Intravitreal brimonidine enhanced the survival and electrophysiological activity of the RGCs in rats. The mechanism of this electrophysiological change may involve activation of the Trk-MAPK/ERK and Trk-PI3K signals.

Purpose: To study the safety profile of Lutein/Zeaxanthin(L/Z)-based natural dye solutions in in vitro and in vivo models., Material and Methods: In vitro cytotoxicity and cellular growth experiments were carried out on ARPE-19 and human corneal epithelial (HCE) cell lines using different L/Z-based dye solutions, either alone or in association with brilliant blue (BB) or trypan blue (TB). Light and transmission electron microscopy studies were performed seven days after intravitreal injection of dye solutions in rabbits. Electroretinogram (ERG) recordings were taken at baseline and before histopathology., Results: In vitro cytotoxicity assays demonstrated that the different L/Z-based solutions (from 0.3 to 2%), either alone or in association with BB (0.025%) or TB (0.04%), did not significantly alter mitochondrial activity (≤15%) in the cell lines tested. In addition, in vitro cell growth was inhibited by up to 60% depending on the dye solution, and in direct proportion to the concentration assayed. There was no evidence of structural alterations in the neurosensory retina, retinal pigment epithelium (RPE), or choriocapillaris-choroidal complex. b-Wave ERG records showed no significant differences (±15.2%) in comparison with baseline., Conclusions: L/Z-based dye solutions demonstrated a safe profile in in vitro and in vivo models, and may be a useful tool for staining intraocular structures.

Aim: To study the effects of intravitreally injected triamcinolone acetonide (TA) and/or its preservative benzyl alcohol (BA) in healthy rabbit retina., Methods: Forty-eight rabbits (aged 4 months, body weight ≈3 kg) were randomized into four groups (n = 12). They were examined with electroretinography (ERG) prior to drug exposure, and then injected intravitreally with a combination of TA and BA, TA without BA, BA alone or a balanced saline solution (BSS). The electroretinograms were assessed 1 week and 7 weeks post-injection. The rabbits were euthanized and the sectioned retinas were studied. Immunohistochemical analysis was performed on rods, cones, rod bipolar cells, horizontal cells, amacrine cells and Müller cells., Results: Rabbits injected with BA showed a significantly lower rod-mediated b-wave amplitude than the controls 1 week after injection. TA-injected rabbits demonstrated significantly higher a- and b-wave amplitudes in the total retinal response than the controls 1 week post-injection. The rabbits injected with TA + BA demonstrated a significantly higher b-wave amplitude in the total retinal response than the controls 1 week after injection. The significantly higher a-wave amplitude in the total retinal response remained in the TA-injected rabbits 7 weeks after injection. Immunohistochemistry revealed that protein kinase C alpha (PKC α) was down-regulated in both the perikarya and the axons of bipolar cells in histological sections from rabbit retina injected with TA + BA, BA and TA., Conclusions: Intravitreal injection of the preservative BA reduces the isolated rod-mediated retinal response in the rabbit, transiently and selectively. Intravitreal injection of TA increases the total retinal response in the rabbit up to seven weeks after injection. The effects observed are not only limited to retinal function, but also include changes in the expression of PKC α in rod bipolar cells, indicating drug-related interference with normal retinal physiology in the healthy rabbit eye.

Purpose: To evaluate the in vivo and in vitro toxicity of sunitinib malate, a multikinase inhibitor molecule., Design: Experimental, Prospective, Controlled., Methods: Human retinal pigment epithelial (ARPE-19) and human umbilical vein endothelialcells (HUVECS) were used in a culture toxicity test and exposed to different concentrations of sunitinib malate for 18 hours. The HUVECs also were cultured to evaluate the angiogenesis inhibitory effect of sunitinib malate. Fundus photography and angiographic, electrophysiologic, and histopathologic evaluations with light and electron microscopy were performed in two groups of five rabbits each that received different intravitreal concentrations of the drug. Each rabbit received 0.1 ml of sunitinib malate in the right eye (one group with 12.5 mg/ml, the other group with 25 mg/ml); all animals received 0.1 ml of physiologic saline solution in the left eye. After sacrifice, the eyes were enucleated and fixed with modified Karnovsky solution., Results: No toxicity related to sunitinib malate was observed using an in vitro model with the 12.5 and 25 mg/ml solutions in HUVEC and ARPE cell cultures. No toxicity was observed in the in vivo model with 12.5 mg/ml, but light microscopy showed that the 25 mg/ml solution damaged the photoreceptors layer. No functional changes in the electroretinogram were observed in any group., Conclusions: Sunitinib malate 12.5 mg/ml caused no toxicity in in vivo and in vitro models, but the 25 mg/ml concentration caused retinal changes suggesting toxicity in the in vivo model. Further research with the drug is needed in models of ocular neovascularization.

Purpose: To compare the intravitreal pharmacokinetic profile of a triamcinolone acetonide formulation containing the preservative benzyl alcohol (TA-BA) versus a preservative-free triamcinolone acetonide formulation (TA-PF), and evaluate potential signs of toxicity to the retina., Methods: A total of 60 New Zealand male white rabbits, divided into two groups, were studied. In the TA-BA group, 30 rabbits received an intravitreal injection of TA-BA (4  mg/0.1 ml) into the right eye. In the TA-PF group, 30 rabbits received an intravitreal injection of TA-PF (4  mg/0.1 ml) into the right eye. The intravitreal drug levels were determined in 25 animals from each group by high-performance liquid chromatography (HPLC). The potential for toxicity associated with the intravitreal triamcinolone injections was evaluated in five randomly selected animals from each group by electroretinography (ERG) and by light microscopy., Results: Median intravitreal concentrations of TA-BA (µg/ml) were 1903.1, 1213.0, 857.8, 442.0, 248.6 at 3, 7, 14, 21 and 28 days after injection. Intravitreal concentrations of TA-PF (µg/ml) were 1032.9, 570.1, 516.6, 347.9, 102.8 at 3, 7, 14, 21 and 28 days after injection. The median intravitreal triamcinolone concentration was significantly higher in the TA-BA compared to the TA-PF group at 7 days post-injection (p < 0.05). There was no significant difference between the two groups in median triamcinolone concentration at the other time points evaluated. There was no evidence of toxic effects on the retina in either group based on ERG or histological analyses., Conclusions: Following a single intravitreal injection, the median concentration of triamcinolone acetonide is significantly higher in the TA-BA compared to the TA-PF group at 7 days post-injection. No toxic reactions in the retina were observed in either group.

Aims: The purpose of this study was to investigate the influence of glutathione on the electroretinogram (ERG) in diabetic and non-diabetic rats., Materials and Methods: Streptozotocin (STZ: 60 mg/kg) was injected into male RCC Wistar rats to induce hyperglycemia, with buffer instead of STZ injected into age-matched non-diabetic controls. After 8 weeks, ERG measurements were obtained at seven different scotopic flash intensities on the two groups of anesthetized, dark-adapted rats (controls, STZ). Following ERG measurements, eyes were enucleated for measurements of retinal/vitreous GSH and glutathione disulfide (GSSG)., Results: Diabetic rats produced delayed b-wave ERG signals (increased implicit times), but had normal a-wave and b-wave amplitudes, a-wave implicit times, and oscillatory potentials. No differences were observed in retinal GSH or GSSG between controls and diabetics; however, correlations between GSH and all ERG parameters (with the exception of b-wave implicit times) were noted, and were not significantly altered by the presence of hyperglycemia., Conclusions: GSH is likely to play an important role in retinal function as assessed by the ERG, with this role not substantially altered in rats diabetic for 8 weeks.

Purpose: To evaluate the intraocular properties of A36, a peptide that directly antagonizes the cell surface urokinase receptor and so prevents pericellular urokinase plasminogen activator activity., Methods: A total of 41 rabbits were used. The toxicity study tested three doses of A36: 1 mg/ eye, 0.3 mg/eye, and 0.1 mg/eye. At 2 and 12 weeks, eyes were evaluated by ERG and histology. Pharmacokinetics were studied in rabbit eyes with the dose of 1 mg/eye in two different formulations: a micronized preparation and a non-micronized formulation. Eyes were enucleated at months 1, 2, 3, 4, and 5. Vitreous, retina, and choroid were collected separately for active A36 analysis., Results: We did not find ocular toxicity with low and medium doses. At the highest dose, there was a transient toxicity at 2 weeks but was not notable at 3 months. The target choroid concentration of A36 was chosen as > or =100 nM. The micronized formulation at months 1, 2, and 3 combined, showed variable levels in the choroid giving 5/10 (50%) of the therapeutic level; the non-micronized formulation at months 4 and 5 combined, gave 6/7 (86%) of the therapeutic level, although this difference was not statistically significant., Conclusion: A36 appears to be long lasting; the non-micronized formulation of A36 gave concentrations above therapeutic level in the choroid at months 4 and 5. Optimization of the formulation of A36, particularly the particle size, may result in a promising new compound for exudative age-related macular degeneration treatment.

Purpose: NiCl(2) (15 microM) enhances the ERG b-wave amplitude of vertebrate retina, up to 1.5-fold by blocking E/R-type voltage-gated Ca(2+) channels, which is mediated by blocking the release of GABA onto ionotropic GABA-A and GABA-C receptors. In vivo, it is likely that zinc, rather than nickel ions, may be involved in the modulation of retinal signalling. Therefore, we tested the effect of both, ZnCl(2) (10 to 500 microM) and DEDTC (100 to 500 microM), which chelates zinc ions for the capacity to influence the ERG b-wave amplitude., Methods: Transretinal potentials from the isolated bovine retina were recorded as electroretinograms and Ca(2+) inward currents by patch-clamp recordings of stably Ca(v)2.3 transfected HEK-293 cells, yielding an IC(50) value of 5.3 microM for ZnCl(2)., Results: ZnCl(2) (10-15 microM) increased the b-wave amplitude by 1.52-fold +/- 0.12 (n = 6 retinas), which was partially reversible upon washout. The same 1.5-fold stimulation of the b-wave amplitude was reported recently for 15 microM NiCl(2). The superfusion of isolated retinas by DEDTC (100 microM) caused a transient decrease of the ERG b-wave amplitude (0.75-fold +/- 0.06; n = 4), suggesting that the co-secretion of Zn(2+) ions may occur under scotopic conditions., Conclusion: The stimulatory effect of ZnCl(2) on the ERG b-wave amplitude resembles the stimulatory effect of NiCl(2) and may be mediated rather by the NiCl(2)-sensitive, Ca(v)2.3 E-/R-type voltage-gated Ca(2+) channels than by NiCl(2)-sensitive T-type channels.

Purpose: To evaluate the short-term toxic effects of pegaptanib sodium on retinal function. At present, intraocular anti-vascular endothelial growth factor (VEGF) therapy is the primary choice of treatment for neovascular maculopathy. The isoform VEGF(165) is specifically inhibited by pegaptanib sodium. Therefore, since VEGF(165) has neuroprotective effects against apoptosis of neuronal cells, blockage of VEGF(165) by pegaptanib could induce retinal dysfunction. In the present study, we used an electrophysiological technique for testing retinal toxicity in order to evaluate the short-term toxic effects of pegaptanib sodium on retinal function in a model of isolated perfused vertebrate retina., Methods: Isolated bovine retinas were perfused with an oxygenated, pre-incubated nutrient solution. Electroretinograms (ERGs) were recorded as trans-retinal potentials using Ag/AgCl electrodes. Pegaptanib sodium (0.006, 0.06, or 0.2 mg/ml) and solvent carrier were added to the nutrient solution for 45 min. ERGs were monitored before, during, and after exposure., Results: No significant reductions of b-wave (p = 0.357, p = 0.31, and p = 0.11, respectively) or a-wave (p = 0.189, p = 0.46, and p = 0.23, respectively) amplitudes were detected during application of pegaptanib (0.006, 0.06, or 0.2 mg/ml). The solvent carrier alone had no effect on ERG b- or a-waves (p = 0.98 and p = 0.42, respectively). During washout, ERG amplitudes of all test series remained unchanged., Conclusion: Results suggest that both pegaptanib sodium and its solvent carrier have good safety profiles. Intraocular application of 0.3 mg pegaptanib sodium induced no significant changes in ERGs in our ex vivo model and, thus, appears to be safe.

Purpose: To investigate the potential toxicity of the vehicle used for triamcinolone acetonide (TA) to the cornea, lens, ciliary body, and retina of pigmented rabbits., Methods: Forty chinchilla rabbits (40 eyes) were divided into four groups: group A (control group) eyes received an intravitreal injection of 0.1 ml sterile saline solution; group B eyes received 0.1 ml (1.3 mg) TA plus vehicle; group C eyes received 0.1 ml (1.3 mg) TA alone (vehicle eliminated); group D eyes received 0.1 ml vehicle only. Intraocular pressure (IOP) was measured pre-injection and at 1, 7, 14, 30, and 90 days post-injection. Scotopic and photopic electroretinograms (ERG) were examined pre-injection and 7, 30, and 90 days post-injection. Animals were sacrificed 7, 30, and 90 days post-injection. Eyes were enucleated and examined by light microscopy (LM) and electron microscopy (EM)., Results: The IOP of groups B and C was higher than that of other groups on days 1, 7, and 14 post-injection (p < 0.05). ERG amplitudes of groups B and D were lower than those of other groups on days 7 and 30 post-injection (p < 0.05). Histopathological sections indicated morphologic changes in the ciliary body, lens, and retina of eyes in groups B and D., Conclusions: Vehicle used for TA is toxic to the lens, ciliary body, and retina of pigmented rabbit eyes after injection of intravitreal TA.

Purpose: To evaluate the ocular safety of varying doses of a single intravitreal injection of the candidate neuroprotective agent, recombinant human erythropoietin (rhEPO)., Methods: Thirty New Zealand rabbits were divided into one of six groups: untreated controls, intravitreal saline injection, and intravitreal injections of rhEPO (100 U, 250 U, 500 U, or 1000 U). Electroretinography (ERG) was performed one day prior to injection and on post-injection days 3, 7, 14, and 21. Fluorescein angiography was done on post-injection day 28 and graded for the presence of neovascularization by a masked observer. Animals were sacrificed for histologic examination 30 days after injection., Results: Except for the rhEPO 500 U group on day 21, there were no statistically significant differences in the amplitude or implicit time of the ERGs between groups or at different timepoints. Fluorescein angiography showed no evidence of neovascularization. Light microscopy showed no apparent abnormalities in retinal morphology or evidence of retinal damage compared to control groups., Conclusion: A single 0.1-ml intravitreal injection of rhEPO at a dose of up to 1000 U does not appear to cause adverse effects on retinal vasculature, retinal anatomy, or ERG function in albino rabbits.

Purpose: To investigate the intraocular properties and toxicity of IMS2186, a small molecule developed as an anti-choroidal neovascularization (anti-CNV) drug., Materials and Methods: Cellular toxicity and mechanism of action was tested on cell lines in vitro. Intraocular studies used rabbits for drug dissolution as well as toxicity and rats for the treatment study as well as the toxicity confirmation study. Rabbits' eyes were injected with 2.5 mg of IMS2186 and observed for 36 weeks. Laser-induced CNV in rats was treated with IMS2186, Kenalog, or phosphate-buffered saline (pBS). Fluorescein angiography (FA) and immunohistochemical processing of the globes was performed., Results: The anti-proliferative IC(50) of IMS2186 for human fibroblast cells was 1.0-3.0 microM and 0.3-3.0 microM for human cancer cells; the IC(50) of IMS2186 to inhibit endothelial tube formation was 0.1-0.3 microM. The IC(50) of IMS2186 for inhibiting the production of pro-inflammatory cytokines was 0.3-1 microM. The IC(50) of IMS2186 for inhibiting macrophage migration was 1 micrM. These biological properties were not species specific. IMS2186 can be formulated as a suspension for long-lasting release and when delivered intraocularly, no intraocular toxicity was observed by slit lamp exam, fundus exam, intraocular pressure measurements, or by electroretinography. FA showed a reduction in the leakage in eyes treated with IMS2186 and triamcinolone acetonide; DAPI staining also showed significantly less cellularity in IMS2186-treated lesions as compared to PBS (p = 0.0025)., Conclusion: IMS2186 may be a safe intraocular therapeutic agent for intraocular proliferation and angiogenesis.

Purpose: Repeated intravitreal injections of endothelin-1 (ET-1) lead to alterations in the visually evoked potentials (VEPs) and loss of retinal ganglion cells (RGCs) in rabbits. The purpose of this study was to determine whether kallidinogenase can offset the alterations induced by ET-1., Methods: ET-1 (2.5 x 10(-7) M, 20 microL) was injected into the vitreous of the right eye of rabbits (ET-1-treated eyes, n = 30) twice a week for 4 weeks. The vehicle for ET-1 was injected into the left eye on the same schedule (vehicle treated eyes, n = 30). During this 4 weeks period, kallidinogenase (1.0 unit/kg/day, kallidinogenase-treated group) or saline (saline-injected control group) was continuously delivered intravenously by an implanted osmotic pump. VEPs were recorded before, and 2 weeks and 4 weeks after, the first ET-1 injection, and all rabbits were sacrificed at 4 weeks. The number of RGC cells was counted in hematoxylin- and eosin-stained retinal sections. In the analyses, the ET-1 induced alterations were normalized to the values in the vehicle treated control eyes, i.e., kallidinogenase (K) + ET-1/K+ vehicle or saline (S) +ET-1/S + vehicle. Retinal sections were also examined by immunohistochemistry with antibodies to single-stranded DNA (ssDNA) or to glial fibrillary acidic protein (GFAP). The effect of kallidinogenase on the ONH blood flow was determined by a hydrogen gas clearance flowmeter., Results: The significant prolongation of the relative VEP implicit times (ITs) 4 weeks after the ET-1 injection (P < 0.01, paired t test; post-ET-1 vs. pre-ET-1) was significantly decreased by kallidinogenase (P < 0.001, t test, K + ET-1/K+ vehicle vs. S +ET-1/S + vehicle). The relative number of RGCs was decreased in the saline-injected group, and this decrease was also decreased by kallidinogenase (P < 0.05, t test, K + ET-1/K+ vehicle vs. S +ET-1/S + vehicle). ssDNA staining showed fewer apoptotic cells in the retina of kallidinogenase-treated rabbits. Intravitreal injection of ET-1 also decreased the blood flow in the optic nerve head and increased the GFAP immunostaining and axonal degeneration. These changes were also counteracted by kallidinogenase., Conclusion: These results indicate that kallidinogenase can counter the effects of ET-1 and should be considered for the treatment of ischemic retinal and optic nerve disorders related to abnormal ET-1 production.

The treatment of vitreoretinal diseases is limited and, nowadays, new drug delivery approaches have been reported in order to increase drug bioavailability. The objective of the current study was to determine the pharmacokinetic profile of a biodegradable dexamethasone acetate implant inserted into the vitreous of rabbits and to evaluate its potential signs of toxicity to the rabbits' eyes. The results showed that the intravitreous drug concentration remained within the therapeutic range along the 8-week period of evaluation. The system under study was not toxic to the normal rabbit retina, and no significant increase in intraocular pressure was observed.

Purpose: To investigate the effects of H-7 and Latrunculin B (Lat-B) on retinal vascular permeability and electrophysiology at concentrations that increase outflow facility in monkeys., Methods: One eye of 1 rhesus and 22 cynomolgus monkeys received an intravitreal bolus injection of H-7 or Lat-B; the opposite eye received vehicle. Multifocal electroretinograms (mfERGs), and photopic and scotopic full-field electroretinograms (ffERGs, sERGs) were recorded in subsets of monkeys at baseline and at multiple time-points post-H-7 or Lat-B. Vitreous fluorophotometry (VF) and fluorescein angiography (FA) were also performed., Results: No differences between the H-7 or Lat-B treated and control eyes were found in ffERGs, mfERGs, sERGs, or in FAs in any monkey. No significant difference was found in vitreous fluorescein levels between H-7 treated or Lat-B treated vs. control eyes., Conclusions: No effect on retinal vascular permeability or retinal electrophysiology was apparent after intravitreal administration of H-7 or Lat-B at doses that increase outflow facility and lower IOP when given intracamerally.

Purpose: To investigate the subretinal toxicity profile of the ribozyme to the proliferating cell nuclear antigen (PCNA-Rz) and 5-fluorouracil (5-FU), as well as the highest nontoxic subretinal dose of the mixture of the two agents in rat eyes., Methods: Brown-Norway rats received subretinal injections of 1 microg, 10 microg, and 100 microg/microl PCNA-Rz and 0.06 microg/microl, 0.3 microg/microl, and 1.5 microg/microl 5-FU in the right eyes, and the left eyes were injected with H-BSS as control. Each dose was tested on 5 eyes in a 5 microl volume. In a second study, a combination of 5-FU (1.5 microg/microL) with varying 10-30-50 microg/microl doses of PCNA-Rz was tested in a regimen of four sequential subretinal injections. Toxicity was monitored by biomicroscopy, indirect ophthalmoscopy, electroretinography (ERG), and histology., Results: The highest nontoxic dose for subretinal PCNA-Rz was 10 microg/microl, whereas 100 microg/microl showed disturbance of pigmentation with corresponding histological changes of retinal photoreceptor loss and retinal pigment epithelium proliferation or irregularities. Subretinal injection of all three doses of 5-FU did not show any toxicity. Serial injections of a mixture of 1.5 microg/microl 5-FU with 10 microg/microl of PCNA-Rz was found to be safe in rat eyes., Conclusions: Subretinal injections of the combination of PCNA-Rz (10 microg/microl) and 5-FU (1.5 microg/microl) demonstrated to be safe in rat eyes during the course of this study, even with a multiple administration of four injections.

Purpose: To investigate the effect of intraocular erucylphosphocholine (ErPC) on the retina, the retinal pigment epithelium (RPE), and the choroid in an in vivo rat model., Methods: Adult male Brown Norway rats were injected intravitreally with ErPC dissolved in balanced salt solution (BSS) at a final concentration of 10 or 100 microM with BSS serving as control. Adverse effects on the anterior and posterior segment were assessed by slit-lamp biomicroscopy and ophthalmoscopy. Retinal toxicity was assessed by electroretinography (ERG), retinal ganglion cell (RGC) quantification, and histology 7 days after intravitreal administration of ErPC., Results: There was neither a statistically significant difference in the clinical examination nor in the ERG waves of treated versus control rats 7 days after intravitreal administration of ErPC. Correspondingly, the number of RGC after BSS injection did not differ significantly from ErPC-injected animals. Histologic sections of the posterior segment of 10 and 100 microM ErPC-injected rats did not show any signs of retinal toxicity. Electron microscopy did not display a difference between the 10 microM and the control group. Only the 100 microM-injected animals showed a discrete irregularity of the Müller cell and the retinal ganglion cell cytoplasm at the ultrastructural level., Conclusions: ErPC can safely be injected into the vitreous of adult rats at a concentration of 10 microM without any retinal toxicity. Even a 10-fold increase in ErPC concentration leads only to a discrete cytoplasmic irregularity of the innermost retinal layers.

Purpose: Neurotrophic factors and neurotrophins are well-known to have neuroprotective efficacy against retinal injury. The aim of this experiment is to investigate the signal transduction pathway of ciliary neurotrophic factor (CNTF) on the upregulation of viability of retinal primary culture and retinal protection against constant light damage in vivo. CNTF is known to enhance the viability of retinal culture and provide protection under constant light exposure conditions, but little is known about how the signal transduction pathways of CNTF affect retina function., Methods: Primary retinal cultures were prepared from 7-day-old Wistar rats. Brain-derived neurotrophic factor (BDNF) (0.1, 1, 10 ng/ml), CNTF (0.1, 1, 10 ng/ml), PD98059 (10, 100, 1000 nM), or LY294002 (10, 100, 1000 nM) was added to these cultures at the time of cell preparation. After 3 days, the percentage of cells surviving was assessed using alamarBlue. For the in vivo experiment, inhibitors for the MAPKK (PD98059, 10 microg/eye) or PI3K (LY294002, 10 microg/eye) pathways were injected into the vitreous together with CNTF (1 microg/eye) 2 days before constant light exposure. Electroretinogram (ERG) analysis was performed to investigate which pathway was used by CNTF., Results: CNTF at 1, 10, or 100 ng/ml enhanced cell viability in retinal cultures. The cell-survival activity of CNTF was blocked by 10 ng/ml LY294002 (Dunnet's test, p < 0.05). In vivo, the neuroprotective activity of CNTF in constant-light conditions was attenuated by 10 microg/eye LY294002 (Dunnet's test, p < 0.05)., Conclusions: These data suggest that CNTF promotes cell survival via the PI3K signaling pathway in vitro and in vivo.

We tested the hypothesis that methylmercury chloride (MMC) caused a selective vulnerability in rat retinal cells during the intact preparation. MMC was injected subcutaneously daily at 3 different doses (0.25, 0.70 or 1.50 mg/kg/day) for 30 days. The electroretinograms under dark- and light-adaptation were recorded before and at 10-day intervals during the treatment period. With the lowest dose of MMC, only the amplitude of the light-adapted (LA) 20 Hz response significantly decreased on Day 30. At the intermediate dose, amplitude reductions were observed on Day 20 for the LA 20 Hz response and dark-adapted (DA) a-wave, while reductions in the LA 2 Hz b-wave and DA b-wave were noted only on Day 30. At the highest dose, these changes occurred earlier during the course of treatment. However, the amplitude of the DA second positive oscillatory potentials and the implicit times of any response components remained unchanged at all dosages. These results suggest that the cones are more sensitive than the rods, bipolar cells and Müller cells to MMC. However, amacrine cells were found to be relatively insensitive. Therefore, each retinal cell was found to have a different vulnerability to MMC.

Purpose: This study investigates the role of insulin in retinal function by examining the effects of insulin on the a- and b-waves of the electroretinogram (ERG) recorded from an in vitro bovine retina eye-cup preparation., Methods: Bovine eyes were enucleated immediately after exsanguination, hemisected to form an eye cup, and transported to the laboratory in oxygenated medium. Eye cups were then transferred to a perfusion system that provided constant superfusion of oxygenated perfusate solution to the retina, enabling it to remain in a functional state in vitro. The ERG was recorded, as the retinal response to photic stimulation, from electrodes mounted within the perfusion system. The effects of insulin on the ERG were investigated by adding insulin to the perfusate solution., Results: Application of insulin to the in vitro retina preparation decreased the amplitudes of the a- and b-waves of the ERG in a dose dependent manner with a maximal effect at doses of 0.1 U/ml and above. These effects were reversible., Conclusion: The reduction of ERG amplitudes may result from the hyperpolarising effect of insulin reported in other tissues. The findings suggest that insulin may have a regulatory role in retinal activity; however extrapolation of these results to the intact organism is dependent on the presence of insulin in retina.

We studied the biodegradation of and the tissue reaction to microspheres of 50:50 poly(D,L-lactic-co-glycolic)acid (PLGA) (viscosity-average MW: 3000 d), injected intravitreous in rabbits. These microspheres are under investigation as injectable devices for intravitreous sustained drug delivery. The rate of intravitreous degradation of PLGA microspheres has not been well documented in the literature. Twenty two pigmented rabbits underwent gas vitrectomy in one eye: 19 eyes received 2.5 mg of PLGA microspheres in 1 ml of balanced salt solution (BSS) and 3 control eyes received 1 ml of BSS only. Slit lamp exam and indirect ophthalmoscopy were performed periodically from day 1 to 6 months after surgery. The eyes were enucleated and studied by light microscopy and immunohistochemistry at various time points. The electroretinogram (ERG) was recorded in a subgroup of rabbits before injection and after 1 and 6 months. The amount of microspheres in the vitreous cavity progressively decreased. At 6 months microspheres were found in 1/4 rabbits at indirect ophthalmoscopy and in 4/4 rabbits histopathologically. A mild localized, non progressive foreign body reaction was observed. The cell reaction was composed mostly of vimentin and glial fibrillary acidic protein positive cells which probably represent glial cells and fibroblasts. The choroid and retina were normal. The ERG showed no abnormalities. No clinical inflammatory signs were observed 4 days postoperatively and thereafter.

The present study was undertaken to determine whether dextromethorphan (DM), a potent N-methyl-D-aspartate antagonist, could attenuate the effects of ischemia on rabbit ERG. Retinal ischemia was induced by increasing intraocular pressure to 120 mm Hg for 30, 60, or 90 min. DM was intravenously administered before ischemia and maintained throughout the entire experimental period. ERGs were recorded prior to, during, and after ischemia. The results indicate that the b-wave hyperresponses and the delays in implicit times induced by 30 min. ischemia were suppressed by the administration of DM. Similar findings were obtained when ischemia lasted for 60 min, except that DM did not improve delayed implicit times, suggesting that cellular injury is still present. ERG changes resulting from 90 min ischemia were not reversed by DM treatment. Effects of DM treatment on a-wave were less prominent. Together, our results further support that DM can to some extent alleviate ischemic injury in the rabbit retina.

We investigated the inhibition of proliferation of human retinal pigment epithelial cells in vitro by the 4-quinolone, ciprofloxacin, and the steroid, dexamethasone. The concentration of ciprofloxacin that inhibited growth by 50% (IC50) was found to be 14.1 micrograms/mL. Growth was 100% inhibited at 83 micrograms/mL. At 166 micrograms/mL, all the cells became completely detached and appeared dead at the end of seven days. The IC50 for dexamethasone in RPE cells was found to be 141 micrograms/mL. A dexamethasone concentration of 1.3 mg/mL inhibited proliferation 100% after five days. When the two drugs were combined, the inhibitory effect was found to be additive; i.e., the IC50 dose of the two drugs in combination inhibited RPE cell proliferation by 75%. A combination of the two drugs was also tested for retinal toxicity in rabbit eyes. An examination of histological sections and electroretinograms showed that a dose of 100 micrograms of ciprofloxacin, alone or in combination with 200 micrograms of dexamethasone in saline, was not toxic to the rabbit retina. These studies indicate that a combination of ciprofloxacin and dexamethasone has the potential for reducing the risk of PVR formation and aiding in the prevention of endophthalmitis.

Povidone-iodine is frequently instilled on to the conjunctival surface prior to intraocular surgery in order to prevent septic endophthalmitis. A small amount of povidone-iodine is inevitably introduced into the eye when it is used in this manner. The toxicity of intravitreal povidone-iodine was assessed in rabbit eyes by injecting 0.1 ml of povidone-iodine in concentrations of 0.05%, 0.5% and 5% into the vitreous cavity. The injected eyes were evaluated by clinical examination, anterior segment and fundus photography, endothelial cell counts, electroretinography and histopathology. Compared to control eyes, no changes were observed in all 6 eyes injected with 0.1 ml of 0.05% povidone-iodine solution. 9 of 10 eyes tolerated a concentration of 0.5% with no detectable adverse changes. One eye developed a temporary mild iritis and mild suppression of the ERG. Intra-retinal hemorrhages, edema, arteriolar narrowing and retinal edema were seen one week following injection. Mild retinal necrosis of the same area was seen on histology. All 4 eyes injected with 5% povidone-iodine developed temporary hypotony and iridocyclitis. A dense cataract developed in all eyes. Full thickness retinal necrosis and a profound lasting reduction in the ERG was produced in all of these eyes. No corneal epithelial or endothelial changes were observed in any eye in this series. Low concentrations of intravitreal povidone-iodine likely to be produced by instillation prior to surgery are tolerated by rabbit eyes. The concentrations tolerated by the studied eyes are near reported bactericidal levels.

Hemicholinium-3 effects on the albino rabbit neural retina have been described, but effects on the retinal pigment epithelium (RPE) have not been closely examined. We have studied retinal morphology and function in Dutch belted (pigmented) rabbits after single intravitreal injections of Hemicholinium-3 or saline. DC electroretinogram recordings show a decrease in a, b, and c-wave amplitudes, with the c-wave affected first. Experiments with sodium iodate show that the early decrease in the c-wave results from a loss of the RPE component of the c-wave, rather than the retinal Slow PIII component. After two days, ophthalmoscopic abnormalities of the fundus are severe in a large area with pigmentary changes. A sharp boundary appears between normally pigmented and depigmented fundus, indicative of a critical threshold for damage. The RPE contains clumped melanin. Pigmented cells are seen away from the basement membrane, an early histological observation temporally correlated with a loss of barrier function seen in fluorescein angiograms. After 10 days, apparent proliferation of non-pigmented RPE cells coincides with re-establishment of the barrier. Rod photoreceptor outer segments are lost 4-7 days after injection in the depigmented regions of the fundus, but outer segments are spared in normally pigmented fundus areas. This regional pattern is distinct from that seen in albino rabbit retina where outer segment loss is fairly uniform. We conclude that Hemicholinium-3 affects the RPE in pigmented rabbits in addition to known effects on retinal cholinergic neurons and photoreceptor disc synthesis.