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C. Nien Shy, M. Chwa, M. Kenney, S. Bose; Triiodo-thyronine (T3) Activates Caspase-3 and Caspase-8 Pathways in Primary Cultured Mouse Extraocular Muscle Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5276.
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Mitochondria are the major site of oxidative phosphorylation in the cell and are considered a likely target for the action of 3,3’,5’ triiodothyronine (T3) in thyroid eye disease; it is hypothesized that T3 induced hyperthyroidism in extraocular muscle cells may result in altered caspase pathway activity which are known activators of apoptosis. We used C57BL/6 wild-type mice to obtain extraocular muscle cells which were treated with different concentrations of T3 and subjected to caspase pathway assays.
Extraocular muscles were isolated from 4-6 weeks old, female wild-type C57BL/6. Tissues were minced, enzymatically digested with trypsin and type V collagenase and plated on tissue culture flasks. Mouse extraocular muscle cells were isolated, cultured and exposed to different concentrations (6,12, 24 and 48ng/ml) of T3 for 7 days. Caspase 3, 8, 9 and 12 were measured by fluorochrome caspase assays using a Fluorescence Image Scanning Unit (FMBIO III) Instrument (Hitachi, Yokohama, Japan). Data were analyzed using GraphPadPrismTM 3.0 version statistics program (GraphPad Software Inc., San Diego, CA).
After 7 days the cells treated with 48ng/ml had increased caspase 8 and caspase 3 activities compared to control cultures (p= 0.01). Cells treated with 12ng/ml and 24ng/ml did not show increased caspase-3 and caspase-8 activity compared to control cells (p=0.8) and caspase 9 was not activated at any concentration of T3.
Our findings suggest that higher concentrations (48ng/ml) of T3 induced hyperthyroidism in culture involving extraocular muscle cells is associated with apoptosis involving caspase 3 and caspase 8 pathways. In contrast the midrange T3 levels did not result in significant apoptotic change. We conclude that T3 induced hyperthyroidism in vitro resulted in apoptotic activity involving the caspase dependent pathways.
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