Effect of Novel Vital Dyes on Outer Blood-Retina Barrier Function in Cultured Human Retinal Pigment Epithelium

Abstract

Purpose: 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·cm²). 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·cm², respectively, compared to 73 ± 44.9 Ω·cm² 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. Conclusion: Novel vital dyes (CVR, M2B and Orange II) caused no outer BRB function alteration.

Acid Violet 17: a New Dye for Chromovitrectomy?

“…Higher concentrations (0.25 mg/ ml, 0.5 mg/ml, 1.0 mg/ml) caused significant elevation of LDH, indicating cell damage. The effect of acid violet 17 on the outer blood-retina-barrier function of the retinal pigment epithelium was examined in an in vitro-model of cultivated embryonic monolayers by immunofluorescence of zonulae occludentes and transepithelial electrical resistance (TER) [6] (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.…”

“…In summary of the available data on preclinical in-vitro and invivo testing of AV17 [5][6][7][8], focusing on the intraocular use during vitreoretinal interventions, it is obvious that tested dye concentrations (0.005 to 1 mg/ml) were below the concentration of acid violet 17 that was CE certified and is no available for human use (1.5 mg/ml, ala purple, ala ® medics, Dornstadt/Germany). In none of the reported test settings the composition of dye solutions was similar to the clinically available formulation of ala purple [5][6][7][8]. Surprisingly, superfused porcine retina tests were performed at 30°C.…”

“…10 seconds) as use by Steel et al [9] was experimentally not evaluated. The published preclinical results [5][6][7][8] focused on different retinal cells potentially susceptible for toxic effects of AV17, includ-ing the retinal pigment epithelium, the outer retinal layers, Müller cells, astrocytes, and ganglion cells. Overall, the results indicate that the threshold of toxic side effects, cell damage, or undesired secondary cell reactions may occur at concentrations much lower than the presently marketed preparation of acid violet-17.…”

“…Text on the abscissa indicates the evaluated parameter. ERG = electrretinography, GC = ganglion cells, MG = microglia, LDH (data from[5][6][7][8] = lactate deghydrogenase, s. i. = single intravitreal injection.…”