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CW Doshna, JH Fortner, JC Pfohl, MD Aleo, MW Tengowski, ME Verdugo; Investigation of the Role of Apoptosis in Drug-induced Cataract Formation . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2377.
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Purpose: Study in vivo and in vitro mechanisms of cataractogenesis by investigating cataractogen-induced apoptosis and alterations in N-cadherin [Ncad] and ß-catenin [ßcat] in lens epithelial cells [LEC]. Methods: In vivo: Sprague-Dawley rat pups were treated with selenite, CJ-12,918, lovastatin [LOV], tamoxifen [TMX] or ciglitazone [CIG] for 5-12 days. Apoptosis was evaluated with TUNEL by fluorescent microscopy in lens capsule-epithelium from treated pups. Ncad and ßcat expression in lens sections from treated pups was evaluated immunohistochemically by confocal microscopy. In vitro: A human LEC line treated with TMX, CJ-12,918 or LOV, was evaluated for 1) Ncad and ßcat expression, or 2) apoptosis by TUNEL, caspase-3 activity [casp3] and acridine orange staining. Results: In vivo: Nuclear opacities in selenite-treated pups and cortical opacities in TMX and CJ-12,918-treated pups were observed by day 5. CJ-12,918-induced opacities progressed without further treatment while there were no changes in lens clarity in LOV- or CIG-treated pups. Ncad and ßcat expression in lens sections was not altered by TMX, CIG, CJ-12,918 or LOV treatment. All lens capsule-epithelial preps showed TUNEL-positive LEC.
TUNEL-positive LEC relative to control: View OriginalDownload SlideView OriginalDownload Slide In vitro: Only TMX treatment increased casp3 and TUNEL labeling in HLEC. Ncad and Bcat are expressed in this HLEC line. However, treatment did not appear to affect expression. Conclusion:TUNEL labeling and Ncad and ßcat expression did not correlate with clinical changes in lens clarity. These results indicate the TUNEL assay is not a suitable independent method to determine cataractogenic potential in vivo or in vitro. Other markers may be involved as well as cellular recovery mechanisms. Further studies using quantitative methods are underway to confirm these findings.
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