Effects of light on retinal pigment epithelial cells, neurosensory retinal cells and Müller cells treated with Brilliant Blue G.

BACKGROUND: The aim of this study is to evaluate the safety profile of Brilliant Blue G (BBG) with and without exposure to light (L) on three different retinal cell lines. METHOD: ARPE-19, R28 and MIO-M1 cells were treated with BBG: 0.125 mg/mL (0.5x clinical concentration), 0.25 mg/mL (1x) or 0.5 mg/mL (2x) with or without surgical illumination of halogen light exposure for 10 min, 15 min or 30 min. Cells were further cultured after 24 h and then analysed for cell viability, late stages of apoptosis and mitochondrial damage associated with early apoptosis using assays that measure trypan blue dye exclusion, increases in caspase-3/7 activity or changes in mitochondrial membrane potential (ΔΨm), respectively. RESULT: All three cell lines that were exposed to BBG in the presence or absence of light exposure for 30 min were found to have cell viability and caspase-3/7 activity levels similar to the untreated cultures. The mitochondrial membrane potential (ΔΨm) was decreased significantly at the 2x + L dose and 2x dose in all three retinal cell lines compared to their respective untreated control cells. At the lower doses of BBG, with or without exposure to light, the ΔΨm values were similar to the untreated control cultures. CONCLUSION: Exposure to BBG dye concentrations that are used clinically (0.125 mg/mL and 0.25 mg/mL) in the presence up to 30 min of surgically equivalent light intensity is safe for retinal cells.

Differential effects of nicotine on retinal and vascular cells in vitro.

The purpose of the current study is to understand the effects of nicotine in human retinal pigment epithelial (ARPE-19), human microvascular endothelial cells (HMVEC) and rat neurosensory retinal (R28) cells. ARPE-19, HMVEC and R28 cell cultures were treated with 10(-2) and 10(-4)M nicotine for 24h. R28 cells were also pre-treated for 4h with ALLN and ALLM (calpain inhibitors) or epicatechin, an antioxidant flavonoid compound. Trypan blue dye exclusion assay, caspase-3/7, LDH activity and DNA laddering assays were performed. With 10(-2)M nicotine treatment, R28 cell cultures showed decreased cell viability that was partially reversed by pre-treatment with the antioxidant epicatechin but not with calpain inhibitors. The DNA ladder assay showed a 200bp banding pattern consistent with apoptosis, however, caspase-3/7 activity was not increased. After treatment with 10(-2)M nicotine, HMVEC cultures showed decreased cell viability and increased LDH activity but no DNA banding patterns or caspase-3/7 activity. ARPE-19 cells showed no change in cell viability or caspase-3/7 activity at any of the nicotine concentrations. We conclude of dissimilar responses to nicotine treatment in three different cell lines. Nicotine was toxic to HMVEC and R28 cell cultures but the ARPE-19 cells were unaffected. In R28 cells, the nicotine effects were through an oxidant pathway that is non-caspase, non-calpain mediated while the HMVEC toxicity was via necrosis. Understanding the mechanisms of cell death may have potential therapeutic implications in the treatment of cigarette smoking related retinal diseases such as age-related macular degeneration (AMD).

Effects of Benzo(e)pyrene on Reactive Oxygen/Nitrogen Species and Inflammatory Cytokines Induction in Human RPE Cells and Attenuation by Mitochondrial-involved Mechanism.

PURPOSE: To identify inhibitors that could effectively lower reactive oxygen/nitrogen species (ROS/RNS), complement and inflammatory cytokine levels induced by Benzo(e)pyrene [B(e)p], an element of cigarette smoke, in human retinal pigment epithelial cells (ARPE-19) in vitro. METHODS: ARPE-19 cells were treated for 24 hours with 200 µM, 100 µM, and 50 µM B(e)p or DMSO (dimethyl sulfoxide)-equivalent concentrations. Some cultures were pre-treated with ROS/RNS inhibitors (NG nitro-L-arginine, inhibits nitric oxide synthase; Apocynin, inhibits NADPH oxidase; Rotenone, inhibits mitochondrial complex I; Antimycin A, inhibits mitochondria complex III) and ROS/RNS levels were measured with a fluorescent H2 DCFDA assay. Multiplex bead arrays were used to measure levels of Interleukin-6 (IL-6), Interleukin-8 (IL-8), Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), Transforming Growth Factor alpha (TGF-α) and Vascular Endothelial Growth Factor (VEGF). IL-6 levels were also measured by an enzyme-linked immunosorbent assay. Real-time qPCR analyses were performed with primers for C3 (component 3), CFH (inhibits complement activation), CD59 (inhibitor of the complement membrane attack complex (MAC)) and CD55/DAF (accelerates decay of target complement target proteins). RESULTS: The ARPE-19 cultures treated with B(e)p showed significantly increased ROS/RNS levels (P < 0.001), which were then partially reversed by 6 µM Antimycin A (19%, P = 0.03), but not affected by the other ROS/RNS inhibitors. The B(e)p treated cultures demonstrated increased levels of IL-6 (33%; P = 0.016) and GM-CSF (29%; P = 0.0001) compared to DMSO-equivalent controls, while the expression levels for components of the complement pathway (C3, CFH, CD59 and CD55/DAF) were not changed. CONCLUSION: The cytotoxic effects of B(e)p include elevated ROS/RNS levels along with pro-inflammatory IL-6 and GM-CSF proteins. Blocking the Qi site of cytochrome c reductase (complex III) with Antimycin A led to partial reduction in B(e)p induced ROS production. Our findings suggest that inhibitors for multiple pathways would be necessary to protect the retinal cells from B(e)p induced toxicity.

Effects of triamcinolone acetonide on human trabecular meshwork cells in vitro.

AIM: To study the effects of triamcinolone acetonide (TA) on cultured human trabecular meshwork (HTM) cells. MATERIALS AND METHODS: HTM cells were cultured and treated with 125, 250, 500 and 1000 µg/mL concentration of TA for 24 h. The cells were treated with both crystalline TA (TA-C) (commercial preparation) and solubilized TA (TA-S). Cell viability was measured by a trypan blue dye exclusion test. The activity of caspse-3/7 was measured by a fluorescence caspase kit and DNA laddering was evaluated by electrophoresis on 3% agarose gel. Levels of lactate dehydrogenase (LDH) were assessed with LDH cytotoxicity assay kit-II. RESULTS: Mean cell viabilities of HTM cells after 24 h exposure to TA-C 125, 250, 500, and 1000 µg/mL were 75.4 ±2.45% (P < 0.0001), 49.43 ± 1.85% (P < 0.0001), 17.07 ± 2.39% (P < 0.0001), and 3.7 ± 0.9% (P < 0.0001), respectively, compared with the untreated HTM cells 92.49 ± 1.21%. The mean cell viabilities with 125, 250, 500, and 1000 µg/mL of TA-S were 94.47 ± 1.60% (P > 0.05), 90.13 ± 0.40% (P < 0.01), 85.57 ± 0.47% (P < 0.001), and 71.67 ± 3.30% (P < 0.0001), respectively, compared to DMSO-equivalent cultures. Untreated HTM control had a cell viability of 96.57 ± 1.98%. DMSO-treated controls of 125, 250, 500, and 1000 µg/mL had a cell viability of 94.73 ± 0.57%, 96.97 ± 1.08%, 93.97 ± 1.85%, and 97.27 ± 1.15%, respectively. There was no increase of caspase-3/7 activity in cultures treated with either TA-C or TA-S. DNA laddering showed no bands in the TA-C or TA-S treated cultures. There were significantly higher LDH release rates at all concentrations of TA-C compared to TA-S. CONCLUSIONS: Results show that the effect of TA-C and TA-S on HTM cells is due to cell death by necrosis at all concentrations except 125 µg/mL of TA-S. Elevated levels of LDH confirmed necrotic cell death. Our study also infers the relative safety of TA-S over TA-C.

Use of intraocular human recombinant tissue plasminogen activator as an adjunct treatment of posterior synechiae in patients with uveitis.

PURPOSE: To evaluate the effect of intracameral injection of small doses of human recombinant tissue plasminogen activator (hr-tPA) as an adjunct to lysing extensive recent-onset posterior synechiae associated with uveitis in the setting of impending pupillary seclusion. METHODS: This is an interventional retrospective case series involving three patients. All patients received an intracameral injection of hr-tPA while on maximum antiinflammatory therapy. Two patients had unilateral acute anterior uveitis with extensive (270°-360°) recent-onset posterior synechiae, while 1 patient had chronic recurrent anterior uveitis complicated by recent and preexisting posterior synechiae. RESULTS: Two patients with acute uveitis had rapid and complete synechiolysis (360°) after an intracameral injection of hr-tPA within 24 hours. One patient with acute reactivation of recurrent uveitis had subtotal (270°) synechiolysis because of incomplete lysis of chronic synechiae. With resolution of inflammation, all patients regained their preuveitis visual acuity. No cataract or glaucoma was reported at 12-month follow-up. CONCLUSION: We evaluated the role of intracameral hr-tPA injections as an adjunct to maximum antiinflammatory therapy. We conclude that a low-dose (3 µg) low-volume (0.05 mL) intracameral injection of hr-tPA in patients with extensive recent-onset posterior synechiae associated with uveitis leads to rapid lysis of synechiae, reducing the risk of pupillary seclusion and associated glaucoma.

The role of perivascular melanophage infiltrates in the conjunctiva in sympathetic ophthalmia.

PURPOSE: To report pathologic changes in the conjunctiva from the exciting eye in a case of sympathetic ophthalmia (SO). METHODS: Report of clinical findings and conjunctival histopathology in a patient with SO. RESULTS: A 50-year-old male developed SO, with unusual peribulbar conjunctival pigmentation in the inciting eye. Histological examination of the conjunctival biopsy revealed perivascular distribution of CD68(+) melanophages that also expressed HLA-DR, suggesting that these macrophages may act as antigen-presenting cells. In addition, increased CD4(+) and CD3(+) lymphocytes were noted in the subconjunctival space when compared to specimens of normal conjunctiva and traumatic uveal prolapse without SO, suggesting T-cell recruitment. CONCLUSIONS: These pathologic findings suggest a possible mechanism by which local antigen processing by subconjunctival melanophages may play a role in the initiation of the complex cell-mediated response seen in SO.

Copper deposition in a variant of multiple myeloma: pathologic changes in the cornea and the lens capsule.

PURPOSE: To report the pathologic changes in the cornea and the lens capsule resulting from copper deposition in a variant of multiple myeloma. METHODS: Case report. RESULTS: Light microscopy of the cornea revealed endothelial cell attenuation with diffuse copper deposition in the central Descemet membrane, which showed thinning, whereas copper banding was seen in the midperipheral and peripheral cornea where the Descemet membrane was normal in thickness. Copper deposition was confirmed by x-ray microanalysis. The anterior lens capsule showed subepithelial copper deposits. Thickening, multilayering, and splitting of the lens capsule were also noted. CONCLUSIONS: We report the pattern of deposition of copper in the Descemet membrane of the cornea and the anterior lens capsule in multiple myeloma, associated with hypercupremia. Descemet membrane thinning and regional differences in copper deposition were noted. Also, thickening and splitting of the lens capsule are a novel observation.

Valganciclovir in the treatment of cytomegalovirus retinitis in HIV-infected patients.

Oral valganciclovir is a new and highly efficacious alternative to the chronic administration of ganciclovir in the treatment of cytomegalovirus (CMV) retinitis in HIV-infected patients. In addition to its excellent bioavailability and favorable pharmacokinetic profile, valganciclovir has also proved cost effective and is the most widely used drug in the armamentarium for the treatment of CMV retinitis. Valganciclovir is a prodrug of ganciclovir, the erstwhile commonly used therapy. In March 2001, the US Food and Drug Administration approved valganciclovir for the induction and maintenance treatment of CMV disease, including CMV retinitis. Valganciclvoir has compared favorably with both oral and intravenous treatments for induction and maintenance therapy with ganciclovir. The reduced pill burden and the ease of oral administration has helped avoid the risks associated with intravenous therapy. The most serious adverse event is neutropenia, which makes the patient susceptible to infections. In the current review, we have compiled all the available evidence-based information on valganciclovir.

Inhibition of apoptosis in human retinal pigment epithelial cells treated with benzo(e)pyrene, a toxic component of cigarette smoke.

PURPOSE: To study the inhibitory effects of some agents or drugs (inhibitors) on benzo(e)pyrene (B(e)P)-induced cell death and apoptosis on human retinal pigment epithelial (ARPE-19) cells in vitro. METHODS: ARPE-19 cells were pretreated with varying concentrations of different classes of inhibitors (calpain, benzyl isothiocyanate [BITC], simvastatin, epicatechin, genistein, resveratrol, and memantine) before B(e)P exposure. Cell viability (CV) was determined by a trypan blue dye-exclusion assay. Caspase-3/7 and caspase-9 activities were measured by fluorochrome assays. The production of reactive oxygen/nitrogen species (ROS/RNS) was measured with 2',7'-dicholorodihydrofluorescein diacetate dye assay. RESULTS: At 30-microM concentrations, the genistein, resveratrol, and memantine inhibitors were able to reverse significantly the loss of cell viability, the activation of caspase-3/7 and caspase-9, and the production of ROS/RNS in ARPE-19 cell cultures. Memantine was the most potent and genistein was the least effective inhibitor in blocking the B(e)P-induced effects. Calpain, BITC, simvastatin, and epicatechin did not reverse the loss of cell viability in B(e)P-treated ARPE-19 cells. As a matter of fact, at the concentrations studied (15, 30, 45 microM), the BITC plus B(e)P-treated cultures showed significantly lower cell viability than the B(e)P-treated culture alone, suggesting BITC-related toxicity. CONCLUSIONS: Genistein, resveratrol, and memantine can reverse the apoptosis and oxidant production generated by B(e)P, a toxic element of smoking. These inhibitors may be beneficial against retinal diseases associated with the loss of RPE cells.

Bimatoprost - a review.

Bimatoprost is a synthetic prostamide analog that is efficacious in the treatment of open-angle glaucoma, ocular hypertension and other forms of glaucoma. It reduces intraocular pressure (IOP) by increasing uveoscleral and trabecular outflow. When used as a 0.03% topical preparation once daily, it demonstrates sustained lowering of IOP of 7 - 8 mmHg over a 24-h period. The drug has been found to be more effective than timolol. In some studies it has shown greater ability to lower IOP when compared with other prostaglandin analogs; whereas in others all three clinically used prostaglandin analogs were found to be equally effective. It shows good IOP reduction when used in combination with other glaucoma medications. A common side effect includes mild conjunctival hyperemia, which is generally reversible. Other side effects include periorbital pigmentation, discomfort, ocular surface hyperemia and skin changes. Pharmacoeconomic data indicate that bimatoprost is cost effective in the treatment of open-angle glaucoma.

Effects of benzo(e)pyrene on the retinal neurosensory cells and human microvascular endothelial cells in vitro.

PURPOSE: To study the effects of benzo(e)pyrene (B(e)P), a toxic component of cigarette smoke, on retinal neurosensory (R28) cells and human microvascular endothelial cells (HMVEC). MATERIALS AND METHODS: R28 cells and HMVEC were treated for 24 hours with 1000, 400, 200, and 100 micro M of B(e)P. Cell viability was measured by dye exclusion assay. Caspase-3/7, -8, -9, and -12 activities were measured by fluorochrome assays. DNA ladder was run on agarose gel, and lactate dehydrogenase (LDH) release rate was evaluated using a LDH cytotoxicity kit II. RESULTS: R28 cells exposed to B(e)P 1000 and 400 micro M showed a decrease in cell viability but not at lower concentrations of 200 and 100 micro M. At 400, 200, and 100 micro M B(e)P, there was an increase in caspase-3/7 and at 200 and 100 microM B(e)P caspase-12 activities. Caspase-8 activity was increased only at 200 micro M B(e)P. Caspase-9 activity was not increased at any concentration. DNA ladder revealed bands at 200 bp intervals at lower concentrations and LDH activity at higher concentrations. HMVEC cultures exposed to B(e)P 1000, 400, and 200 micro M showed a decrease in cell viability. Caspase-3/7 activity was not increased at any concentration. DNA laddering revealed no bands at 200 bp intervals at any dose. LDH release rates increased at all three concentrations. However, 100 micro M B(e)P was found safe on HMVEC. CONCLUSIONS: B(e)P has different mechanisms of action on R28 cells and HMVEC at different concentrations. In R28 cells, 200 and 100 micro M of B(e)P causes activation of caspase-3/7, -8 (200 micro M only) and -12 pathways, leading to apoptotic cell death, but, at higher concentrations, there is non-apoptotic cell death, which could be due to necrosis. In contrast, the HMVEC cell death is through non-caspase-dependent necrosis pathway. The molecular mechanisms of cell death vary with different cell types and concentrations of B(e)P.

Aberrant wound-healing response in mitomycin C-treated leaking blebs: a histopathologic study.

OBJECTIVE: To characterize histopathologic features of leaking mitomycin C-treated blebs and aberrant wound healing that may lead to persistent conjunctival thinning and leakage. METHODS: Forty mitomycin C-treated filtering blebs excised for persistent leaks from 40 patients were examined histopathologically using hematoxylin-eosin, periodic acid-Schiff, Masson trichrome, and Alcian blue histochemical stains. RESULTS: Ninety percent of the leaking blebs contained epithelial-stromal domes with areas of acellular stroma covered by attenuated epithelium. Seventy-five percent of the blebs demonstrated varying degrees of fibrovascular repair growing from the bleb margin, either beneath or interdigitating with the acellular zone. A novel observation in 65% of specimens was Alcian blue-positive myxoid stroma at the interface between the fibrovascular proliferation and the epithelial-stromal dome. The association between the presence of fibrovascular proliferation and Alcian blue-staining myxoid stroma was significant by Fisher exact test (P = .002). CONCLUSIONS: A desirable filtration bleb requires a sufficient reparative fibrovascular response to maintain an epithelial-stromal barrier to prevent leakage. Fibroblasts must lay down a continuous collagen-rich fibrous layer, rather than merely myxoid stroma, beneath the conjunctival epithelium to promote bleb stability. Surgical techniques and postsurgical care should aim to attain this desired outcome.