title: Effect of novel vital dyes on outer blood-retina barrier function in cultured human retinal pigment epithelium.
abstract: OBJECTIVE
  To assess tight junction (TJ) integrity in cultured human fetal retinal pigment epithelium (HFRPE) after exposure to clinically relevant novel vital dyes.
  
  
  METHODS
  HFRPE floater cells were harvested from RPE primary cultures of 4 donor eyes and seeded on polyester Transwell® for 4-6 weeks. The apical compartments of well-differentiated cultures were exposed to 0.005 mg/ml Coomassie violet R200 (CVR), methyl 2B (M2B) or Orange II. Periods of 30-300 s were chosen to mimic surgical exposure times, while 3 h was used for toxicity assays, with subsequent washout. Cell-cell junctions were studied by immunofluorescence (zonula occludens-1, ZO-1). Transepithelial electrical resistance (TER) was measured regarding blood-retina barrier (BRB) function.
  
  
  RESULTS
  At 4-6 weeks after confluence, HFRPE had grown into pigmented hexagonal monolayers with stable TER values (451-1,520 Ω·cm(2)). After 300-second dye treatments, a continuous ZO-1 signal was detected in all vital dye-treated groups 1.5 h after exposure, whereas trypsin controls showed patchy loss of the TJ stain. TER of CVR-, M2B- and Orange-II-treated groups had dropped 1.5 h after exposure to 148 ± 58.4, 162 ± 23.7 and 164 ± 18.5 Ω·cm(2), respectively, compared to 73 ± 44.9 Ω·cm(2) in positive controls. After 3 h of exposure to 0.005 mg/ml vital dyes in thick drops, TER maintained similar levels to those prior to exposure (90.8 ± 4.7% of the original values, 93.8 ± 6.5 and 91.9 ± 3.6%, respectively), together with no difference from the vehicle controls (94.8 ± 6.6%). TER values recovered in all groups to prior levels within 3 days.
  
  
  CONCLUSIONS
  Novel vital dyes (CVR, M2B and Orange II) caused no outer BRB function alteration.
authors.affiliation: Department of Ophthalmology, University of Bonn, Bonn, Germany.
authors.forename: Zengping
authors.initials: Z
authors.lastname: Liu
authors.forename: Carsten H
authors.initials: CH
authors.lastname: Meyer
authors.forename: Boris V
authors.initials: BV
authors.lastname: Stanzel
mesh_terms: D001391:Azo Compounds / Q000633:toxicity; D001557:Benzenesulfonates / Q000633:toxicity; D001813:Blood-Retinal Barrier / Q000187:drug effects*; D002478:Cells, Cultured; D004396:Coloring Agents / Q000633:toxicity*; D017097:Electric Impedance; D019084:Fluorescent Antibody Technique, Indirect; D005840:Gentian Violet / Q000633:toxicity; D006801:Humans; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D012394:Rosaniline Dyes / Q000633:toxicity; D019108:Tight Junctions; D014821:Vitrectomy; D062826:Zonula Occludens-1 Protein / Q000378:metabolism
chemical_list: D001391:Azo Compounds; D001557:Benzenesulfonates; D004396:Coloring Agents; D012394:Rosaniline Dyes; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; C024757:Coomassie brilliant blue R; D005840:Gentian Violet; C014722:2-naphthol orange