Abstract
Purpose: :
Human corneal endothelial cells (HCECs) are non mitotic cells. Any intracameral applications of drugs needs evaluation of the potential apoptotic and toxic effect of the used substances. This study was undertaken to investigate the toxicity of recombinant tissue plasminogen activator (rTPA) on cultured HCECs to determine its safety for clinical use.
Methods: :
Cell cultures of HCECs were harvested from human donor eyes and exposed to various concentrations of rTPA (10-200 µg/ml). For cytotoxicity testing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and the Live/DeadTM Viability/Cytotoxicity assay testing were performed. Annexin V binding combined with propidium iodide (PI) co-staining was used for the distinction of viable, early and late apoptotic cells. Odds Ratios (ORs) and confidence intervals (CIs) were calculated for 50 µg/ml, 100 µg/ml and 200 µg/ml. Cell morphology was studied after 24h of exposure to rTPA to identify cellular damage. Immunolocalization of zonula occludens 1 (ZO1) was performed to analyze intercellular barrier disturbance in presence of rTPA.
Results: :
Reduction of mitochondrial dehydrogenase activity following rTPA exposure was dose dependent and suggested a comparable toxicity as the data obtained with the Life/DeadTM assay. The mean number of annexin V-FITC and PI positive cells was not significantly increased at concentrations of 50 µg/ml and 100 µg/ml. For 200 µg/ml, however, the ORs were 5.08 ± 0.48 (95%CI, 3.96-6.20; p = 0.002) for apoptosis and 6.15 ± 0.47 (95%CI, 5.12-7.18; p = 0.008) for necrosis. Increasing concentrations of rTPA resulted in a fading immunopositive staining for ZO1.
Conclusions: :
These data suggest a dose dependent toxic effect of rTPA on HCECs in vitro. While intracameral rTPA concentrations of up to 100 µg/ml seem to be clinically safe, the use of concentrations higher than 125 µg/ml might induce irreversible cell death and should be restricted to selected cases.
Keywords: cornea: endothelium • drug toxicity/drug effects • anterior chamber