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      Hypothermia Protects Cultured Human Retinal Pigment Epithelial Cells against Trypan Blue Toxicity

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          Purpose: To determine whether trypan blue (TB) is toxic to cultured human retinal pigment epithelial (ARPE-19) cells, and whether hypothermia can protect ARPE-19 cells against TB toxicity. Methods: ARPE-19 cells were cultured and exposed to balanced salt solution as a control, while other cells were exposed to 0.05, 0.2, and 0.5% TB dye at 37 and 4°C for 5 and 30 min. The percentage of ARPE-19 cells that survived was determined by resazurin 1 day after the exposure. Results: A statistically significant decrease in the percentage of ARPE-19 cells surviving was found after exposure to 0.2 and 0.5% TB at any temperature or for any exposure duration (p < 0.01). The percentage of RPE cells surviving at 0.05% was not significantly different from that of controls except for a 30-min exposure at 37°C. The percentage of cells surviving at 4°C for a 5-min exposure to 0.5% TB and a 30-min exposure to 0.2 and 0.5% was significantly higher than that at 37°C under each condition (p < 0.01 for all). Conclusions: These results indicate that TB is toxic to human RPE cells, and the toxicity is dose- and exposure duration-dependent. Exposing the cells at 4°C had a protective effect against higher concentrations or longer exposure durations of TB compared to exposure at 37°C.

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          Most cited references 19

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          Trypan blue staining in vitreoretinal surgery.

          To evaluate the efficacy of trypan blue for staining the internal limiting membrane (ILM) and epiretinal membranes (ERM) in vitreoretinal surgery.
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            Trypan blue: effect on retinal pigment epithelial and neurosensory retinal cells.

            To evaluate the toxicity of trypan blue on retinal cells in vitro. Human retinal pigment epithelial cells (ARPE-19) and rat neurosensory retinal cells (R28) were grown in tissue culture and treated with four different concentrations of trypan blue (0.1%, 0.05%, 0.025%, and 0.0125%), in combination with surgical light exposure (0, 5, or 10 minutes). Cell viability, mitochondrial function, and DNA synthesis were measured by trypan blue dye-exclusion assay, mitochondrial dehydrogenase assay, and tritiated [3H] thymidine incorporation, respectively. ARPE-19 and R28 cells exposed to trypan blue with or without illumination did not show any significant decrease, either in cell viability by the dye-exclusion assay or in [3H] thymidine incorporation. R28 cells exposed to 0.1% trypan blue with and without light showed a significant reduction of mitochondrial dehydrogenase activity (P <0.05). ARPE-19 cells exposed to trypan blue, with or without light, did not show any significant decrease in mitochondrial dehydrogenase activity. This study suggests that rat neurosensory retina (R28) cells are more sensitive than human RPE (ARPE-19) cells to trypan blue. ARPE-19 cells showed no evidence of toxicity with any of the three assays, but R28 cells showed evidence of toxicity with the mitochondrial dehydrogenase assay at the higher doses and light-exposure times studied. Clinical studies must be conducted to determine the safety and efficacy of staining of the inner limiting membrane with trypan blue.
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              Functional outcome after trypan blue-assisted vitrectomy for macular pucker: a prospective, randomized, comparative trial.

              To evaluate functional outcome after the intraoperative application of 0.06% trypan blue during vitrectomy for macular pucker. Prospective, randomized, comparative study. Forty-three eyes of 43 consecutive patients, 30 women and 13 men, were incorporated in the study. Patients were randomized into two groups: group 1 (n = 22) with trypan blue (0.06%) staining, and group 2 (n = 21) without trypan blue staining. Functional outcome (best-corrected visual acuity, Goldmann perimetry) was evaluated 1 day before surgery, at 6 weeks, and at intervals of 3, 6, and 12 or more months postoperatively. Only patients with an idiopathic macular pucker were included. In all patients, a standard three-port pars plana vitrectomy with peeling of an epiretinal membrane (ERM) and the internal limiting membrane was performed. No other modification of the surgical procedure, except the use of trypan blue was made between the two groups. Mean age was 68.9 years in group 1 (with trypan blue) and 69.4 in group 2 (without trypan blue). Median best-corrected visual acuity was 20/63 in both groups (range, 20/200-20/40; P >.5) before surgery. Mean follow-up time was 5.8 months in group 1 and 5.3 months in group 2. After surgery, median visual acuity had increased to 20/40 in both groups (range, 20/500 to 20/20 in group 1 and 20/100 to 20/20 in group 2; P .5). An improvement of visual acuity (gain of 2 or more lines) was seen in 16 patients of both groups. No postoperative visual field defects were noted. Trypan blue-assisted vitrectomy for macular pucker leads to good functional results with no dye-related adverse effects after short follow-up. Trypan blue might be especially applicable in cases in which the borders of the ERM are difficult to define. Hypothetical advantages, such as fewer recurrences of ERMs after trypan blue staining, will have to be evaluated during further follow-up of patients.

                Author and article information

                S. Karger AG
                February 2006
                17 February 2006
                : 220
                : 2
                : 114-117
                aDepartment of Ophthalmology and Visual Science, and bDivision of Clinical Cell Therapy, Tohoku University Graduate School of Medicine, and cDepartment of Biofunctional Science, Tohoku University Biomedical Engineering Research Organization, Sendai, and dDepartment of Ophthalmology, Ishinomaki Red Cross Hospital, Miyagi, Japan
                90576 Ophthalmologica 2006;220:114–117
                © 2006 S. Karger AG, Basel

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                Figures: 2, References: 23, Pages: 4
                Original Paper


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