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G.R. Welsandt, H. Mietz, M. Becker, G.K. Krieglstein; Effect of Multiple Expositions of 5–FU and Mitomycin on 3T3 Cells in vitro: Proliferation and Migration Assays . Invest. Ophthalmol. Vis. Sci. 2006;47(13):39.
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Wound healing following trabeculectomy is one of the most important factors that distinguish between a surgical success or failure. The main cells involved are episcleral and Tenon's fibroblasts. Current clinical concepts mostly involve antifibroblastic substances given once (mitomycin) or multiple times (5–Fluorouracil, 5–FU) during or following surgery. So far, only the antiproliferative effect of a single application on cell proliferation in–vitro has been investigated. In a clinical setting, it may become important to apply such substances more than once to the same eye to enhance the clinical effect. For this reason, we investigated the cytotoxic effect of two consecutive applications of 5–FU and /or mitomycin on 3T3 mouse fibroblasts.
3T3 mouse fibroblasts were cultured. First, Mitomycin and 5–Fluorouracil were applied for 5 minutes. Then, the cells were allowed to grow for 24h. The concentrations of the drugs used led to a reduction of cell survival of about 20% at 24h. After that time, the cells were again exposed for another 5 minutes to Mitomycin or 5–Fluorouracil. The cell survival was determined after another 24h using the crystal violet assay. For control cell survival was determined at 48h after single time exposure. In addition, cell migration was examined using the scratch assay at similar time intervals.
Primary cell proliferation was reliably inhibited with concentrations of 62 µg/ml of Mitomycin and 130 µg/ml of 5–Fluorouracil. At 48h, the cell reduction was 41.0% for Mitomycin and 47.5% for 5–FU. Consecutive applications led to a cell reduction of 40.0% (Mitomycin) and 48.6% (5–FU). Combined applications of Mitomycin and 5–FU led to cell reductions around 58 – 61% (p<0.03; Mann–Whitney test). Migration assays had similar results.
The results of this experiment suggest, that the effect of a second application of a 5–FU and mitomycin alone is not effective after a short time period, but the use of more than one substance may lead to a stronger antiproliferative effect. These results should be tested in an animal model next.
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