Effects of Triamcinolone Acetonide on Vessels of the Posterior Segment of the Eye

Abstract

Purpose: : To investigates the effects of Triamcinolone Acetonide (TA) on bovine retinal endothelial cells (BRECs) in vitro and explores the potential vascular toxic effect of TA injected into the vitreous cavity of rats.

Methods: : Subconfluent BRECs were treated with either 0.1 or 1 mg/mL TA in 1% ethanol. Cell viability was evaluated at 24 h,72 h and 5D using MTT and lactate dehydrogenase (LDH) assays. Cell proliferation was evaluated by BrdU test. Apoptosis was evaluated by TUNEL assay, annexin-binding and caspase 3 activation. Caspase-independent cell deaths were investigated by immunohistochemistry using antibodies against apoptosis inducing factor (AIF), Microtubule-associated protein-light chain 3 (MAP-LC3) and LEI/L-DNase II. In vivo, semi-thin and ultrathin structure analysis and vascular casts were performed to examine TA induced changes of the choroidal vasculature. In addition, outer segments phagocytosis assay on primary RPE cells was performed to assess COX-2 and VEGF mRNAs up-regulations with or without TA.

Results: : The inhibitory effect of TA on cell proliferation could not explain the significant reduction in cell viability. Indeed, TA induces a time-dependent reduction of BRECs viability. Annexin-binding positive cells were observed. L-DNase II was found translocated to the nucleus, meaning that LEI was changed into L-DNase II. AIF was found nuclearized in some cells. Positive LC3-labelled cells were not observed. No autophagy or caspase dependent apoptosis was identified. Thereof, while 1mg/mL TA induces mostly necrosis, exposure to lower concentration for 3 to 5 D induces caspase independent apoptosis involving AIF and LEI/L-DNase II. Semi-thin and ultrathin structure analysis and vascular casts revealed that TA mostly affected the choroidal vasculature with a reduction of choroidal thickness and increased the avascular areas of the choriocapillaries. Experiments performed on primary RPE cells showed that TA down-regulates the basal expression of COX-2 and VEGF and inhibits the OS-dependent COX-2 induction but not the OS-dependant VEGF induction.

Conclusions: : This study demonstrates that TA exert direct toxic effect on BRECs through caspase-independent cell death mechanisms. The choroidal changes observed after TA intravitreous injection may have important implications regarding the safety profile of TA use in human eyes.

Keywords: retinal culture • apoptosis/cell death • choroid