Retinal Pigmented Epithelial Cells Cytotoxicity and Apoptosis through Activation of the Mitochondrial Intrinsic Pathway: Role Of Indocyanine Green, Brilliant Blue and Implications for Chromovitrectomy

Abstract

PurposeTo investigate the in vitro effect of four vital dyes on toxicity and apoptosis in a human retinal pigment epithelial (RPE) cell line.MethodsARPE-19 cells were exposed to brilliant blue (BriB), methyl blue (MetB), acid violet (AcV) and indocyanine green (ICG). Balanced salt solution was used as control. Five different concentrations of each dye (1, 0.5, 0.25, 0.05 and 0.005 mg/mL) and two exposure times (3 and 30 min) were tested. Cell viability was determined by cell count and MTS assay and cell toxicity by LDH assay. Real-time PCR and Western blotting were used to access the apoptosis process.ResultsICG significantly reduced cell viability after 3 minutes of exposure at all concentrations (p<0.01). BriB was safe at concentrations up to 0.25 mg/mL and MetB at concentrations up to 0.5 mg/mL, while AcV was safe up to 0.05 mg/ml, after 3 minutes of exposure. Toxicity was higher, when the cells were treated for 30 minutes. Expression of Bax, cytochrome c and caspase-9 was upregulated at the mRNA and protein level after ICG exposure, while Bcl-2 was downregulated. AcV and MetB were similar to control. However, BriB resulted in upregulation of Bcl-2, an antiapoptotic protein.ConclusionsThe safest dye used on RPE cells was MetB followed by BriB and AcV. ICG was toxic at all concentrations and exposure times tested. Moreover, ICG was the only dye that induced apoptosis in ARPE-19 cells. BriB significantly increased Bcl-2 protein levels, which might protect against the apoptosis process.

HIV-1 Tat-Mediated Apoptosis in Human Blood-Retinal Barrier-Associated Cells

“…Human retinal pigment epithelial cells (ARPE-19) were purchased from the American Type Culture Collection (ATCC, Manassas, VA) [19], and D407 cells were obtained from the Central Laboratory of Central South University Xiangy. Both cell lines were cultured in DMEM/F12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen).…”

Acid Violet 17: a New Dye for Chromovitrectomy?

“…Retinal pigment epithelial cell toxicity of acid violet 17 was examined by a cell-viability assay using a standardized immortalized human cell line (ARPE 19) [7] (l " Fig. 2).…”

“…On the other hand, the staining capacity and intensity of these dyes, which guide the surgeon to the ILM, are by far not ideal. Recently an interesting new dye, acid violet 17 (AV17) became commercially available after basic preclinical testing [5][6][7][8] and CE certification. AV17 (synonymous: Coomassie violet R200) is a triarylmethane dye (total formula: C 41 H 44 N 3 O 6 S 2 , molar weight: 761.92 g/mol, structural formula in l " Fig.…”

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“…По данным литературы низкие концентрации 0,01-0,05 мг/мл ИЦЗ и время экспозиции не более 0,5-1 минуты оказывают слабое повреждающее действие на структуры сетчатки [24]. Однако рядом исследователей было продемонстрировано, что интравитреальное использование данного красителя может оказывать повреждающее действие на структуры заднего сегмента глаза [15,24,37]. ИЦЗ проявляет токсические свойства и способен вызвать повреждение фоторецепторов сетчатки, нарушение целостности клеточных мембран, атрофию клеток пигментного эпителия сетчатки (ПЭС), приводя к снижению остроты зрения [20,32].…”