Safety Profile of Lutein- Versus Triamcinolone Acetonide-Based Vitreous Staining.

Purpose: To assess the safety profile of a new lutein-based vitreous dye (LB-VD) formulation compared with various triamcinolone acetonide (TA) formulations with and without subsequent exposure to perfluorodecalin (PFD) in vitro. Methods: Human adult retinal pigment epithelial cells (ARPE-19) were treated with the following formulations: undiluted preserved TA (TA-BA), diluted preserved TA (D-TA-BA), preservative-free TA (TA-PF), and LB-VD. First, cell tolerability was evaluated with MTT, LDH, and ATPlite assays after 1, 5, and 30 minutes of exposure to each tested formulation. Then, cells were sequentially exposed to formulations and PFD. After 24 hours of exposure to PFD, cell tolerability was evaluated through MTT and ATPlite assays. Results: Among the formulations tested, LB-VD showed the highest levels of cell viability, cell metabolism, and cell proliferation and induced the lowest release of LDH, whereas the TA-based formulations demonstrated a cytotoxic effect on ARPE-19 cells in vitro. After subsequent 24-hour exposure to PFD, a greater reduction of cell viability was noted for all the formulations; however, this reduction was not significant only for the combination LB-VD-PFD, which was the best tolerated condition. Conclusions: LB-VD showed a better safety profile compared with all TA-based formulations, even when used in combination with PFD. Translational Relevance: In surgical practice, LB-VD may be preferred to TA-based formulations for vitreous staining in the light of its more favorable safety profile.

Ex vivo evaluation of retinal cytotoxicity after the use of multiple medical devices in pars plana vitrectomy in porcine eyes.

This study aimed to evaluate viability of retinal cells after the use of multiple intraoperative devices, namely a vitreal dye (triamcinolone acetonide,TA), a ERM/ILM dye (solution of trypan blue 0.15% and brilliant blue 0.025%), and two intraocular tamponades, namely perfluoro-n-octane, (PFO) and silicone oil (SO 1000 cSt), with minimal and maximal removal of their residues, during a simulated pars plana vitrectomy (PPV) in porcine eyes ex-vivo. The in vitro cytotoxicity of each of these compounds was verified on ARPE-19 cells by direct tests according to the ISO 10993-5 (2009). Pars plana vitrectomy was performed on 25 enucleated porcine eyes divided in five groups according to the following conditions: Group A) No surgery control: eye bulbs were kept at room temperature for 40 min; Group B) Sham surgery: PPV with the sole use of BSS for 40 min; Group C) Cytotoxic control: PPV with BSS infusion (20 min) followed by intravitreal injection of 1H-PFO (contact time: 20 min); Group D) Surgery with residues: PPV with BSS infusion and sequential intravitreal injection of TA, ERM/ILM dye, PFO and SO, with minimal removal of each compound after a specified contact-time (overall duration: 40 min); Group E) Surgery with minimal residues: PPV performed as in group D, but with maximal removal of each compound (overall duration: 40 min). All the experimental procedures were performed at room temperature. Immediately after surgery, the retina was extracted from each eye bulb and samples of 3-mm diameter were prepared. Retinal viability was determined for each sample by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay. A cell viability <70% was considered the cytotoxicity threshold. Kruskal-Wallis test was used to evaluate the differences in retinal viability between groups. No cytotoxicity was detected in retinal samples in groups A, B and E. Samples from eye bulbs that had undergone surgery with minimal removal of residues (group D) and cytotoxic controls (group C) showed high retinal cytotoxicity. The tested conditions indicated that the combined use of TA, ERM/ILM dye, PFO and SO during PPV does not affect retinal cells viability if all the devices are properly removed, whereas the cytotoxicity detected in group D may suggest that the presence and accumulation of the residues of the compounds used intraoperatively could negatively impact retinal viability due to a cumulative and/or synergistic cytotoxic effect between them, supporting the crucial role of an optimal removal of the intraoperative medical devices to ensure a safe vitrectomy to the patient.

Triamcinolone acetonide-chitosan coated liposomes efficiently treated retinal edema as eye drops.

Macular edema (ME), which is present in various retinal diseases, leads to permanent retinal structural damage and threatens vision. The intravitreal/periocular injection of triamcinolone acetonide (TA) can improve the prognosis of ME; however, further exploration of noninvasive delivery systems is essential. Therefore, as a continuation of our previous study using TA-chitosan coated liposomes (TA-CHLs) as a topical drug delivery system, the present study aimed to determine the drug safety, stability, permeability, and bioavailability of TA-CHLs. The study was based on detecting the delivery of a fluorescent dye to the retina using optical coherence tomography angiography in rats. Marked cellular uptake was observed in cell lines. TA-CHL toxicity was investigated in cell culture. Clinical ocular safety was evaluated by measuring the corneal thickness and intraocular pressure. In preclinical studies on a laser-induced retinal edema rat model, the TA-CHL eye drops had dramatic therapeutic effect in remission of retinal edema over 10 days. These results demonstrated that TA-CHL was nontoxic and had good bioavailability in vitro and in vivo. The results of the present study indicated that this formulation could be an effective therapeutic approach and the TA-CHL eye drops may represent a new option for retinal diseases.

Clear, Aqueous Topical Drop of Triamcinolone Acetonide.

The objective of this study was to develop a clear aqueous mixed nanomicellar formulation (NMF) of triamcinolone acetonide (TA) with a combination of nonionic surfactant hydrogenated castor oil 60 (HCO-60) and octoxynol-40 (Oc-40). In order to delineate the effects of drug-polymer interactions on entrapment efficiency (EE), loading efficiency (LE), and critical micellar concentration (CMC), a design of experiment (DOE) was performed to optimize the formulation. In this study, full-factorial design has been used with HCO-60 and OC-40 as independent variables. All formulations were prepared following solvent evaporation and film rehydration method, characterized with size, polydispersity, shape, morphology, EE, LE, and CMC. A specific blend of HCO-60 and Oc-40 at a particular wt% ratio (5:1.5) produced highest drug EE, LE, and smallest CMC (0.0216 wt%). Solubility of TA in NMF improved 20 times relative to normal aqueous solubility. Qualitative 1H NMR studies confirmed the absence of free drug in the outer aqueous NMF medium. Moreover, TA-loaded NMF appeared to be highly stable and well tolerated on human corneal epithelial cells (HCEC) and human retinal pigment epithelial cells (D407 cells). Overall, these studies suggest that TA in NMF is safe and suitable for human topical ocular drop application.

The effects of commonly used intravitreal steroids on proliferation index of ciliary body-derived mesenchymal stem cells: an in vitro study.

AIM: To investigate the effects of commonly used intravitreal steroids on survival and proliferation (namely, proliferation index) of ciliary body-derived mesenchymal stem cells (CB-MSC). METHODS: CB-MSCs were isolated from newborn rats' eye, and they were expanded in the medium. Commonly used intravitreal steroids such as dexamethasone (Dex) and triamcinolone acetonide (TA) were added into the medium at commonly used concentration in clinical practice (0.1 mg/mL) and at lower concentration (0.01 mg/mL). Proliferation indexes of CB-MSCs were analyzed with the xCELLigence system at nine consecutive times (at 3rd, 6th, 21th, 30th, 45th, 60th, 75th, 90th and 100th h). RESULTS: Both TA and Dex at both 0.01 mg/mL and 0.1 mg/mL concentrations had negative effect on proliferation indexes of CB-MSC. Although negative effect of TA on proliferation index of CB-MSC at both concentrations was not statistically significant, statistically significant negative effect of Dex at 0.01 mg/mL concentration started 60th h (p = 0.017) and 0.1 mg/mL concentration started 30th h (p = 0.014). DISCUSSION: Even therapeutic doses of intravitreal corticosteroid agents might have negative effects on limited numbers of stem cells. Especially, Dex caused statistically significant toxic effects on CB-MSCs even at lower concentrations of those used clinically. These novel findings deserve further in vivo investigations.

Lidocaine potentiates the deleterious effects of triamcinolone acetonide on tenocytes.

BACKGROUND: Local anesthetics are commonly used for the treatment of a variety of tendinopathies in combination with corticosteroids injection. The goal of this study was to evaluate the effects of lidocaine and triamcinolone acetonide (TA) on cultured rat tenocytes and to determine whether there is a synergistic effect. MATERIAL/METHODS: Rat patellar tendon-derived tenocytes were cultured with or without TA and lidocaine, and the culture without any additive served as the control. Cell morphology and cell viability were evaluated. Expressions of tenocyte-related genes were measured by qRT-PCR. RESULTS: TA, when exposed to tenocytes in vitro, significantly decreased cell viability. The cells cultured with TA had a flattened shape. Moreover, the expressions of tenocyte-related genes in tenocytes were markedly decreased in the TA-treated group. We found that 1% lidocaine synergistically increased the deleterious effects of TA. CONCLUSIONS: Our data provide evidence of the detrimental effects of these drugs on tendon tissues. Injection of TA in combination with 1% lidocaine should be used with caution.

Pharmacokinetics and retinal toxicity of various doses of intravitreal triamcinolone acetonide in rabbits.

PURPOSE: To compare the pharmacokinetics and retinal toxicity of various doses of intravitreal triamcinolone acetonide (TA) in rabbits. METHODS: The rabbits received intravitreal injections of 4 mg and 8 mg TA. The drug concentrations were determined with high-performance liquid chromatography after extraction from the vitreous at various time points. The main pharmacokinetics parameters were calculated with 3p97 pharmacokinetics software. The intraocular pressure, electroretinography, and pathological examinations were evaluated before and after intravitreal injection of different doses of TA. RESULTS: The half-life of intravitreal injection of 4 mg and 8 mg TA was 24 days and 34 days, respectively. No significant differences were found in intraocular pressure (p>0.05) and the electroretinography b-wave amplitudes (p>0.05) among the rabbits before and after intravitreal injection of 4 mg and 8 mg TA. Light and electron microscopy did not show any retinal damage in any group. CONCLUSIONS: Intravitreal injection of 4 mg and 8 mg TA are safe for the rabbit retina. The injection of 8 mg TA produced a longer vitreous half-life and had a prolonged effect on the retina. This conclusion may be referenced in the clinical application of TA in retinal diseases.

Protective effects of N-acetylcysteine on triamcinolone acetonide-induced lens damage in rats.

OBJECTIVE: To examine the relationship of cataract forming effect of intravitreal triamcinolone acetonide (IVTA) injection with oxidative status and the effect of N-acetylcysteine (NAC) on these alterations. MATERIALS AND METHODS: Twenty-six Wistar-Albino rats were included in the study. Rats were assigned into four groups as follows: intravitreal saline injection group (controls); IVTA injection group; IVTA + intraperitoneal NAC injection group (IVTA + NAC); and intraperitoneal NAC injection group (NAC). Triamcinolone acetonide was intravitreally injected at a dose of 1 mg. NAC was intraperitoneally injected at a dose of 150 µg/g body weight. Animals were sacrificed and lens specimens were analyzed for levels of malondialdehyde (MDA) and protein carbonyl (PC) and activities of glutathione (GSH) and glutathione peroxidase (GSH-Px). RESULTS: We found that the MDA and PC levels of lenses were increased in the IVTA group (p < 0.01). It was seen that GSH and GSH-Px in lenses were decreased in the IVTA group (p < 0.01). NAC administration significantly ameliorated these changes in the IVTA + NAC group (p < 0.05). CONCLUSION: These results indicate that the NAC produces a protective mechanism against IVTA-induced cataract and suggest a role of oxidative stress in pathogenesis.

Platelet-rich plasma protects rotator cuff-derived cells from the deleterious effects of triamcinolone acetonide.

Triamcinolone acetonide (TA) injections are widely used to treat enthesopathy, but they may induce adverse effects such as tendon impairment and rupture. Platelet-rich plasma (PRP) is a blood fraction containing high platelet concentrations and various growth factors that play a role in tissue repair processes. The purpose of this study is to investigate whether TA has deleterious effects on human rotator cuff-derived cells, and if PRP can protect these cells from the effects of TA. Human rotator cuff-derived cells were cultured with and without TA and PRP, and the culture without any additive served as the control. Cell morphology was assessed at days 7 and 21. Cell viability was evaluated at days 1, 7, 14, and 21 by a water-soluble tetrazolium salt assay. Induction of apoptosis was measured by immunofluorescence staining and flow cytometry at day 7. Induction of cleaved caspase-3 was measured by immunofluorescence staining at day 7. The cells cultured with TA had a flattened and polygonal shape at day 7. The cells cultured with both TA and PRP were similar in appearance to control cells. Exposure to TA also significantly decreased cell viability, but cell viability did not decrease when PRP was added along with TA. The number of apoptotic cells increased with TA exposure, while addition of PRP prevented cell apoptosis. In conclusion, the deleterious effect of TA was prevented by PRP, which can be used as a protective agent for patients receiving local TA injections.

Triamcinolone acetonide decreases outflow facility in C57BL/6 mouse eyes.

PURPOSE: To determine the effect of triamcinolone acetonide (TA) on outflow facility in mice. METHODS: Animals received 20 µL of TA (40 mg/mL) suspension subconjunctivally either bilaterally or unilaterally and were euthanized after either 1 week or 3 weeks. Before mice were killed, IOP was measured with a rebound tonometer. Outflow facility was determined using simultaneous pressure and flow measurements. Another set of animals received bilateral injection of anecortave acetate (AA) with or without bilateral TA injection and their outflow facility was also determined. Myocilin expression was investigated in a subset of eyes using quantitative PCR (qPCR). RESULTS: Outflow facility of eyes in animals receiving bilateral TA injection (TA(BL)) and TA-treated eyes of animals receiving unilateral injection (TA(UL)) was significantly decreased compared to naïve control eyes (C(naive)) after 1 week and 3 weeks of TA treatment (ANOVA P < 0.01, P < 0.001, respectively). Eyes treated with AA (with or without TA) had higher outflow facility than animals treated with TA (P < 0.05). IOP data did not show any significant difference between groups. qPCR analysis revealed significant decrease in myocilin expression in eyes receiving AA compared to naïve control and TA-treated eyes (ANOVA P < 0.001). CONCLUSIONS: Steroid treatment significantly decreases outflow facility in C57BL/6 mice despite having small effect on IOP. This animal model can be useful for studying the pathogenesis of steroid-induced glaucoma.

Further characterization of ocular safety profile of commercially available preserved and preservative-free triamcinolone acetonide.

PURPOSE: To characterize the safety profile of triamcinolone acetonide (TA) for intraocular application. METHODS: In vitro cell viability assay was performed on 2 types of human ocular cells to evaluate the cytotoxicity of the simulated different vitreal concentrations (from a 1:15 dilution as if injected into 1.5 mL of rabbit vitreous to 1:50 dilution as if injected into 5 mL of human vitreous) of preservative and excipient (supernatant) from Kenalog-40. In vivo 35 guinea pigs were used for evaluating either a dose of intravitreal triamcinolone acetonide (Kenalog-40 and Triesence) or the supernatant of Kenalog-40. The animal eyes were monitored by biomicroscopy, ophthalmoscopy, tonometry, electroretinography, and histology. RESULTS: A ≥ 1:15 dilution of triamcinolone acetonide supernatant from Kenalog-40 did not show cytotoxicity on cultured human pigment epithelial (retinal pigment epithelium) cells or Müller cells. In vivo, neither intravitreal 6 µL (0.248 mg, equivalent to 4 mg in 0.1 mL for human eyes) nor 18 µL (0.744 mg, equivalent to 4 mg in 0.1 mL for rabbit eyes and equivalent to 12 mg in 0.1 mL for human eyes) of triamcinolone acetonide suspension showed ocular toxicity. No significant difference was noted between Kenalog-40 and Triesence clinically and histopathologically. CONCLUSION: The equivalent triamcinolone acetonide doses to 0.1 mL (4 or 12 mg) intravitreal injection for human eye were found safe in guinea pig eyes. No significant difference was noted for 0.1 mL intravitreal injection between Kenalog-40 and Triesence.

Toxicity profiles of subretinal indocyanine green, Brilliant Blue G, and triamcinolone acetonide: a comparative study.

BACKGROUND: This study introduces a novel porcine model to examine the histopathological and electrophysiological consequences of retinotoxicity exerted by dyes commonly used for internal limiting membrane (ILM) staining. METHODS: Indocyanine green (ICG) 0.5 mg/ml, Brilliant Blue G (BBG) 0.25 mg/ml and triamcinolone acetonide (TA) 13 mg/ml was injected subretinally in 12 vitrectomized pig eyes. At 6 weeks, retinas were examined by multifocal electroretinography (mfERG), ophthalmoscopy, fluorescein angiograpy, histopathology, and apoptosis assay. RESULTS: mfERG responses were significantly lower in ICG-injected eyes than in healthy fellow eyes (p = 0.039). The ratio between injected eyes and healthy fellow eyes was lower in the ICG group than in the BBG (p = 0.009) and TA group (p = 0.025). No difference between BBG and TA existed. All retinas were reattached, and fluorescein angiographies showed a window defect corresponding to the injected areas but no blood-retina barrier break-down. Histopathology confirmed damage to the outer retina after ICG, but not after BBG and TA. No apoptosis was found at 6 weeks. CONCLUSIONS: Subretinal ICG induces histological and functional damage to the retina, suggesting that ICG should be used with caution in macular hole surgery, where subretinal migration can occur. In contrast, BBG and TA appear safe after subretinal injection.

Preservative-free triamcinolone acetonide injectable suspension versus "traditional" triamcinolone preparations: impact of aggregate size on retinal biocompatibility.

PURPOSE: To evaluate the biocompatibility of the three currently most commonly used triamcinolone acetonide (TA) preparations on retinal cells. METHODS: Preservative containing KL (Kenalog-40; Bristol-Myers Squibb, Princeton, NJ), compounded preservative-free triamcinolone acetonide (PFTA; compounded from Volon A; Dermapharm, Vienna, Austria), and preservative-free triamcinolone acetonide injectable suspension (TRIESENCE; Alcon, Inc, Fort Worth, TX) (0.01-1 mg/mL) were either added directly on top or separated by a Boyden chamber filter or by a layer of vitreous to confluent cell cultures of retinal pigment epithelial cells (ARPE19) or retinal ganglion cells (RGC5). The distribution pattern of the TA crystals was assessed microscopically. Cell viability was assessed using MTT-ELISA and Live/Dead-Assay. RESULTS: Sedimentation of triamcinolone acetonide injectable suspension, KL, or PFTA caused a pronounced decrease in cell viability. Cytotoxicity was most pronounced when triamcinolone acetonide injectable suspension and PFTA were used. Without direct sedimentation of TA crystals on top of the cells, none of the three formulations were cytotoxic. Triamcinolone acetonide injectable suspension showed the largest and most dense TA crystal aggregates on top of the cells. CONCLUSION: Retinal cytotoxicity of TA seems only to occur when there is intimate contact of TA crystals with the cellular membrane. Cytotoxicity depends on the number and size of TA crystal aggregates-with larger conglomerates being more harmful. Of the TA formulations tested, triamcinolone acetonide injectable suspension had the strongest tendency to form large TA crystal conglomerates and to gravitate downward.

Evaluation of the toxicity of triamcinolone acetonide and dexamethasone sodium phosphate on human lens epithelial cells (HLE B-3).

PURPOSE: The purpose of this study was to compare the in vitro effects of triamcinolone acetonide (TA) and dexamethasone sodium phosphate (DEX) on human lens epithelial cells (HLE B-3). METHODS: HLE B-3 cells were exposed for 24 h to commercially available TA (c-TA) and dimethylsulfoxide-solubilized TA (s-TA). The cells were treated with 1,000 (clinical dose), 750, 500, 200, and 100 µg/mL concentrations of c-TA, s-TA, and supernatant for 24 h. The cells were also treated with DEX at 2, 1, 0.5, 0.2, 0.1 (clinical dose), and 0.05 mg/mL. Cell viability, caspase-3/7 activity, and DNA fragmentation analyses were performed. RESULTS: The mean cell viabilities of HLE B-3 after exposure to c-TA at 1,000, 750, 500, 200, and 100 µg/mL were significantly reduced compared with control untreated cells. The s-TA also significantly reduced cell viability at 1,000, 750, and 500 µg/mL compared with dimethylsulfoxide control. The supernatant did not reduce cell viability. Caspase-3/7 activity significantly increased after treatment with c-TA and s-TA. DNA laddering revealed bands at 200 bp intervals with both c-TA at≥100 µg/mL and s-TA at ≥500 µg/mL. The cell viabilities of HLE B-3 after 24 h exposure to DEX were significantly reduced at 2 and 1 mg/mL but not at lower concentrations tested. Caspase-3/7 activities in HLE B-3 cells were not increased significantly after treatment with DEX at any dose tested. DNA laddering did not reveal any band at any dose tested. CONCLUSION: This study showed that TA at its clinical dose (1,000 µg/mL) in both commercial preparation and solubilized forms decrease HLE B-3 cell viability through an apoptotic pathway. DEX at its clinical dose (0.1 mg/mL) does not decrease cell viability or cause any increase of caspase-3/7 activity. This study suggests that for long-term sustained-release devices, DEX may be less damaging to human lens cells than TA.

Intravitreal triamcinolone acetonide induced changes in the anterior segment in a pig model of branch retinal vein occlusion.

BACKGROUND: Triamcinolone acetonide (TA) has applications for the treatment of a large range of intraocular vascular diseases. The present study in pigs was performed to investigate histopathological and histochemical changes in the levels of myocilin deposition in the anterior segment in a model of branch retinal vein occlusion (BRVO) after vitreal administration of TA. METHODS: After ophthalmoscopic examination, intraocular pressure (IOP) measurement and fundus photography, a BRVO was created photothrombotically in each eye of six pigs, using argon green photocoagulation. The left eye was then injected intravitreally with 4 mg/0.1 ml TA. After 11 weeks, the eyes were re-examined, animals sacrificed, and eyes enucleated and processed in paraffin and epoxy resin. Immunofluorescence cytochemistry on paraffin sections was performed to localise the distribution of myocilin in the anterior segment and histology by light and transmission electron microscopy on epoxy resin sections on TA-treated and untreated eyes. RESULTS: Histology revealed pathological changes in the TA-treated eye, including swollen mitochondria, layered long endoplasmic reticulum, pleomorphic nuclei, dense fibrillar extracelluar deposits and aggregates of unusual cell inclusions. Myocilin levels were significantly higher in the TA-treated eyes in the trabecular meshwork (p = 0.001), ciliary process (p = 0.011) and iris (p = 0.030) than in the untreated eyes. CONCLUSIONS: This study suggests that increased myocilin synthesis and related ultrastructural changes in the anterior segment after treatment with intravitreal TA in a porcine model of retinal oedema in BRVO may contribute to IOP elevation.

Investigation of polyethylenimine-grafted-triamcinolone acetonide as nucleus-targeting gene delivery systems.

BACKGROUND: Nuclear membrane is one of the main barriers in polymer mediated intracellular gene delivery. To improve the transgenic activity and safety of nonviral vector, triamcinolone acetonide (TA) as a nuclear localization signal was conjugated with different molecular weight polyethylenimine (PEI). METHODS: Different molecular weight PEI [600, 1800, 25,000 (25k)] was conjugated with TA to synthesize PEI-TA by two-step reaction. Their physicochemical characteristics, in vitro cytotoxicity and transfection efficiency were evaluated. To investigate the difference of transfection efficiency of various molecular weight PEI-TA, their transfection mechanism was further investigated by confocal microscopy and competition assay. Transgenic expression in vivo was evaluated by injection into hepatic portal vein of mice. RESULTS: All PEI-TA could form nanosize polyplexes with DNA and their physicochemical properties resemble each other. Their cytotoxicities were negligible compared to PEI 25k. The order of transfection efficiency was PEI 1800-TA > PEI 600-TA > PEI 25k-TA. A transfection mechanism study displayed that TA could inhibit considerably the transgenic activity of PEI 1800-TA and PEI 600-TA, but that of PEI 25k-TA was not inhibited. It was suggested that PEI 1800-TA and PEI 600-TA might translocate into the nucleus. Confocal microscopy investigation verified this suggestion. The data strongly suggested that the transfection efficiency of PEI 1800-TA in vivo was much higher than that of PEI 25k, which was consistent with the results obtained in vitro. CONCLUSIONS: Low molecular weight PEI-TA could translocate into the nucleus efficiently. PEI 1800-TA presented higher transgenic activity and it has a great potential for gene therapy as a nonviral carrier.

Functional and structural effect of intravitreal indocyanine green, triamcinolone acetonide, trypan blue, and brilliant blue g on rat retina.

PURPOSE: The purpose of this study was to evaluate the functional and structural damage of the retina after intravitreal injections of four different dyes in the rat. METHODS: Rats were injected intravitreally with indocyanine green (ICG), trypan blue, triamcinolone acetonide, or brilliant blue G in the right eye. The other eye was injected with saline and served as a control. Simultaneous bilateral electroretinograms were recorded before injection and 7 and 28 days after injection. Histology and immunohistochemistry analyses with antibodies recognizing glial fibrillary acidic protein and protein kinase C were performed 28 days after the initial injection on both eyes. RESULTS: Seven days after dye injection, the electroretinogram response of the treated eyes was altered in each group. At 1 month, eyes injected with triamcinolone acetonide, trypan blue, or brilliant blue G fully recovered, whereas eyes treated with ICG had A-wave and B-wave reduction of 65% and 63%, respectively. The inner nuclear layer thickness was statistically decreased in the ICG group (P = 0.003) but not with other dyes. Protein kinase C staining was decreased in the ICG group only, but no abnormal qualitative staining was found with either glial fibrillary acidic protein or protein kinase C antibodies with any dye. CONCLUSION: Among the four tested dyes, only ICG led to functional and structural retinal damage.

Electrophysiological retinal pigment epithelium changes observed with indocyanine green, trypan blue and triamcinolone.

BACKGROUND: Intravitreal application of indocyanine green (ICG), trypan blue (TB) or triamcinolone (TA) during vitreoretinal surgery has been associated with severe damage to the retinal pigment epithelium (RPE). However, the physiological background of these findings remains to be assessed. METHODS: In bovine RPE choroid preparations maintained in Ussing chambers, the effect of apical application of ICG, TA (filtered and not filtered) and TB at different concentrations was evaluated. The electrophysiological parameters (transepithelial potential, tissular resistance and short-circuit current) were continuously monitored. The experiments were conducted either in daylight or in dark-adapted conditions. RESULTS: After apical application of ICG and TA (purified and not purified), all bioelectrical parameters were affected in a dose-dependent manner. No significant changes were observed when the preparations were exposed to daylight. No changes were observed with TB. CONCLUSIONS: The electrophysiological results of apical application may explain the toxic effects observed after intraoperative use of ICG and TA. In this RPE study, TB appears to be the safest visualization aid for vitreoretinal surgery.

Treatment of sheep steroid-induced ocular hypertension with a glucocorticoid-inducible MMP1 gene therapy virus.

PURPOSE: To investigate whether intracameral injection of the adenovirus vector AdhGRE.MMP1 would reduce or prevent elevated intraocular pressure (IOP) induced by corticosteroids in living animals. METHODS: Glucocorticoid-inducible adenovirus vectors carrying wild-type or mutant forms of human metalloproteinase 1 (MMP1 and mutMMP1) cDNAs were generated. An adenovirus carrying no gene (Ad5.CMV.Null) was used as an additional control. Sheep were injected intracamerally with 30 microL of each vector, either previously or after the induction of increased IOP with topical prednisolone or sub-Tenon triamcinolone under various protocols. IOP was measured with a Perkins tonometer. Inflammation was monitored by visual inspection. RESULTS: In eyes in which IOP was already elevated to 24 to 30 mm Hg, injection of AdhGRE.MMP1 reduced IOP by 70% in 24 hours and to 10 to 13 mm Hg in 48 hours. In eyes with normal IOP (9-11 mm Hg), preinjection of the virus protected against the increase in IOP normally produced by the corticosteroid. IOP remained at a level of approximately 12 mm Hg for 5 days despite the continuous application of the corticosteroid. Injections of the control viruses had no hypotensive effects. There were no signs of ocular inflammation or discomfort to the animals. CONCLUSIONS: A single dose of a gene therapy vector carrying an inducible metalloproteinase human gene can both protect against the IOP increase produced by corticosteroid instillation in the sheep model and quickly reverse the IOP increase previously elicited by the corticosteroid. These results are a first step toward a treatment of steroid-glaucoma with inducible overexpression of extracellular matrix modulator genes.

Effects of triamcinolone acetonide on vessels of the posterior segment of the eye.

PURPOSE: This study investigates the effects of triamcinolone acetonide (TA) on retinal endothelial cells in vitro and explores the potential vascular toxic effect of TA injected into the vitreous cavity of rats in vivo. METHODS: Subconfluent endothelial cells were treated with either 0.1 mg/ml or 1 mg/ml TA in 1% ethanol. Control cells were either untreated or exposed to 1% ethanol. Cell viability was evaluated at 24 h, 72 h, and five days using the tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5 phenyltetrazolium bromide test (MTT) and lactate dehydrogenase (LDH) assays. Cell proliferation was evaluated by 5-bromo-2-deoxyuridine (BrdU) test. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL assay), annexin-binding, and caspase 3 activation. Caspase-independent cell deaths were investigated by immunohistochemistry using antibodies against apoptosis inducing factor (AIF), cytochrome C, microtubule-associated protein (MAP)-light chain 3 (MAP-LC3), and Leukocyte Elastase Inhibitor/Leukocyte Elastase Inhibitor-derived DNase II (LEI/L-DNase II). In vivo, semithin and ultrathin structure analysis and vascular casts were performed to examine TA-induced changes of the choroidal vasculature. In addition, outer segments phagocytosis assay on primary retinal pigment epithelium (RPE) cells was performed to assess cyclooxygenase (COX-2) and vascular endothelial growth factor (VEGF) mRNAs upregulation with or without TA. RESULTS: The inhibitory effect of TA on cell proliferation could not explain the significant reduction in cell viability. Indeed, TA induced a time-dependent reduction of bovine retinal endothelial cells viability. Annexin-binding positive cells were observed. Cytochrome C was not released from mitochondria. L-DNase II was found translocated to the nucleus, meaning that LEI was changed into L-DNase II. AIF was found nuclearized in some cells. LC3 labeling showed the absence of autophagic vesicles. No autophagy or caspase dependent apoptosis was identified. At 1 mg/ml TA induced necrosis while exposure to lower concentrations for 3 to 5 days induced caspase independent apoptosis involving AIF and LEI/L-DNase II. In vivo, semithin and ultrathin structure analysis and vascular casts revealed that TA mostly affected the choroidal vasculature with a reduction of choroidal thickness and increased the avascular areas of the choriocapillaries. Experiments performed on primary RPE cells showed that TA downregulates the basal expression of COX-2 and VEGF and inhibits the outer segments (OS)-dependent COX-2 induction but not the OS-dependent VEGF induction. CONCLUSIONS: This study demonstrates for the first time that glucocorticoids exert direct toxic effect on endothelial cells through caspase-independent cell death mechanisms. The choroidal changes observed after TA intravitreous injection may have important implications regarding the safety profile of TA use in human eyes.