Abstract
Purpose: :
To study the potential toxicity of triamcinolone acetonide (TA) on retinal pigment epithelial cell line(ARPE–19) in vitro and in the rat eye in vivo.
Methods: :
Sub confluent ARPE–19 cells were treated with 0.1 or 1mg/ml Triamcinolone acetonide (TA), (Sigma Saint–Quentin Fallavier, France). Cell viability was evaluated at 24 and 48 hours using MTT assay and Trypan blue exclusion test. Apoptosis was evaluated by TUNEL assay and annexin–binding. Immunohistochemistry was performed with anti–apoptosis inducing factor (AIF), cytochrome C, and LEI/L–Dnase II antibodies. Nuclei morphology was analyzed with DAPI staining. Western–blot analysis was performed for activated caspase–3, phosphorylated and non–phosphorylated JNK. Finally, cytoplasmic vacuoles were labeled with monodansylcadaverin. For in vivo experiments, 50µg of TA were injected in the vitreous of Lewis rat eyes. Histological analysis of the RPE was performed 8 days later.
Results: :
In vitro, TA formed dense aggregates that were toxic to ARPE–19 cells. Addition of TA previously solubilized in ethanol to the RPE cultures induced a dose–dependent cell death while addition of ethanol alone did not induce significant cell death. No sign of apoptosis were detected either by TUNEL assay, annexin binding, nuclei staining or activated–caspase 3 on western–blot. The alternative LEI/Dnase II pathway (caspase independent) was not activated. However, intense vacuolization positive for monodansyl cadaverine along with phosphorylation of JNK were detected. In vivo, ultra–thin RPE morphology, studied trough serial ultra thin sections, 8 days after TA injection, showed RPE degeneration with optically empty vacuoles.
Conclusions: :
In vitro and in vivo observations demonstrate that TA induce a non–apoptotic, non necrotic RPE cell death. These findings may have important implications regarding the safety profile of TA use in human eyes.
Keywords: apoptosis/cell death • retinal pigment epithelium • corticosteroids