Purchase this article with an account.
Zengping Liu, Carsten H. Meyer, Boris V. Stanzel; Effect Of Chromovitrectomy Dyes On Outer Blood Retinal Barrier Function In Cultured Human Retinal Pigment Epithelium (RPE). Invest. Ophthalmol. Vis. Sci. 2012;53(14):2635.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To assess tight junction (TJ) integrity in cultured fetal RPE after exposure to novel chromovitrectomy dyes by transepithelial resistance (TER) measurements and immunofluorescence microscopy.
Fetal human RPE were obtained from 3 donors and cultured on PET inserts for 6-8 weeks. They were then exposed to Indocyanine green (ICG) (PULSION, Germany) (0.125 mg/ml, 0.05 mg/ml, 0.025 mg/ml) and two novel dyes Coomassie Violet R200 (CVR) (0.005 mg/ml), Methyl 2B (M2B) (0.005 mg/ml) both provided by Fluoron®. The exposure time for ICG was 180 seconds, while it ranged from 30- 300 seconds for CVR and M2B. Balanced salt solution with Ca++/ Mg++ or solvent provided by Fluoron® served as vehicle controls, and 0.5% trypsin/0.2mM EDTA as the positive control. TER was assessed by a voltohmeter. Culture morphology was monitored by phase contrast microscopy. Post exposure distribution of the tight junction protein ZO-1 was studied by immunofluorescence.
At 6-8 weeks post confluence, the RPE had grown into pigmented, hexagonal monolayers with donor-dependent TER values ranging from 600 to 1300 ohm*cm2, suggesting good TJ function. Following exposure to above dyes, TER values were lowest at 1.5h post exposure (158 ± 39.6 ohm*cm2), and back to prior levels after 3 days. Compared to vehicle control, ICG treatment groups showed significant decrease in TER values at 1.5h and 3h post exposure, while no significant decrease existed in CVR and M2B treatment groups. Positive controls showed a significant drop in TER at 1.5h post exposure (71 ± 38.8 ohm*cm2) and remained significantly decreased compared to other groups. Cell morphology remained unchanged with ICG and CVR exposure compared to vehicle control, while there was patchy cell-cell dissociation in positive controls. A hexagonal and continuous ZO-1 signal was detected at apical intercellular junctions in 0.125 mg/ml ICG group and vehicle controls at 1.5h post exposure, whereas trypsin-treated controls showed discontinuous and/or patchy loss of TJ staining
ICG exposure induced a transient decrease in TER values, but appeared not break down TJ integrity in preliminary experiments. Thus, CVR and M2B may be safer dyes in chromovitrectomy surgery with regard to outer BRB function.
This PDF is available to Subscribers Only