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B. D. Kuppermann, A. Jayaprakash Patil, A. Sharma, M. F. Estrago Franco, S. Mansoor, V. Raymond, M. C. Kenney; Effects of Triamcinolone Acetonide on Human Trabecular Meshwork Cells in vitro. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1614.
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© ARVO (1962-2015); The Authors (2016-present)
To study the effects of triamcinolone acetonide (TA) (Kenalog; Bristol-Meyers Squibb, Princeton, NJ) on human trabecular meshwork (HTM) cells.
HTM cells (courtesy of Vincent Raymond) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. The cells were treated with either crystalline TA (TA-C) (commercial preparation) or solubilised TA (TA-S). TA-S was prepared by centrifuging TA-C at 5000 rpm for one minute and then removing the supernatant. We solubilised the residual pellet in DMSO equivalent to the amount of removed supernatant. Cells were treated with 125, 250, 500 and 1000 µg/mL concentration of either TA-C or TA-S for 24 hours. Cell viability was measured by a trypan blue dye exclusion test. To evaluate for apoptosis, activity of caspase-3/7 was measured by fluorescence caspase kits and DNA laddering was performed by electrophoresis on 3 % agarose gel.
The mean cell viability of HTM cells after 24 hours exposure to TA-C 125, 250, 500 and 1000 µg/mL was 75.4%±2.45 (P<0.001), 49.43%±1.85 (P<0.001), 17.07%±2.39(P<0.001), 3.7%±0.9(P<0.001) respectively compared with the untreated HTM cells 92.49%±1.21. The mean cell viability with 125, 250, 500 and 1000 µg/mL of TA-S at 24 hours was 94.47%±1.6(P>0.05), 90.13%±0.4(P<0.01), 85.57%±0.47(P<0.001), 71.67%±3.3(P<0.001) respectively compared with the 1000 µg/mL equivalent DMSO treated control 96.57%±1.9. There was no caspase-3/7 activity with either TA-C or TA-S at any dose. DNA laddering showed no bands with either TA-C or TA-S at any dose.
These results show that both TA-C and TA-S are toxic to HTM cells at all concentrations with the exception of lowest concentration of TA-S (125 µg/mL). As there is no caspase-3/7 activity with either TA-C or TA-S and as we could not find any bands on DNA ladder, we conclude that the toxicity of TA-C and TA-S on HTM cells is due to necrosis at all concentrations except 125 µg/mL of TA-S.
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