A. Jayaprakash Patil

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 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 μ 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 μ M showed a decrease in cell viability but not at lower concentrations of 200 and 100 μ M. At 400, 200, and 100 μ M B(e)P, there was an increase in caspase-3/7 and at 200 and 100 μM B(e)P caspase-12 activities. Caspase-8 activity was increased only at 200 μ 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 μ 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 μ 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 μ M of B(e)P causes activation of caspase-3/7, −8 (200 μ 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.

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

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.

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.