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N. C. Wade, I. Grierson, L. Paraoan, A. F. Clark; A Bovine Model for the Further Study of Cross Linked Actin Networks (CLANs). Invest. Ophthalmol. Vis. Sci. 2007;48(13):2070.
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The cytoskeleton in trabecular meshwork cells plays a key role in the normal outflow of aqueous humour from the eye. Cytoskeletal changes such as cross linked actin networks (CLANs) have previously been identified as a possible indicator of glaucomatous change in organ cultured human trabecular meshwork (TM) tissue and in human TM cells in cultures treated with the synthetic steroid Dexamethasone (Dex). It has been shown that the bovine eye is highly steroid responsive1. We wanted to test whether BMW cells produced large numbers of CLANs and could therefore serve as a useful model.
BMW cells, and the human cell lines (TM5, TM3) and NTM were cultured for 2 weeks in the presence of 10-7 M Dex. Similarly, bovine eyes were dissected and TM tissue was peeled out, placed into a 6 well plate and floated in DMEM plus 10-7 M Dex. Cells and tissues were fixed with 10% NBF and stained with Alexa Fluor 488 Phalloidin to demonstrate actin and counterstained with the nuclear stain Propidium Iodide. Images were taken using our confocal microscope and the percentage of CLAN containing cells was determined by counting numbers of CLANs per nuclei in both confocal assistant and aequitas image analysis programs.For experiments with aqueous humour, we collected 1ml of aqueous per bovine eye within 3 hours of death. This pooled aqueous was stored at -80C prior to use.
BMW cells produced more CLANs than any of the human cell lines tested. After 2 weeks we found that 50% + 3.93 % of BMW cells had CLANs compared to 23% + 3.28 % in the most reactive human cell line (TM3). Following DEX withdrawal the half life for CLANs was 2.4 days in BMW cells and 3.7 days in human cells. We demonstrated that CLANs are also found in whole bovine TM tissue following exposure to Dex. Additionally, we found that BMW cells treated with 100% aqueous humour developed more CLANs (14.1% + 0.69%) than cells treated with normal cell culture medium (3.39% + 0.35%).
We have shown that the bovine eye and BMW cells are useful models for the further study of CLANs due to the high numbers of CLANs produced following DEX exposure. A population of 50% CLANs should make functional and biochemical studies less problematic. We have also shown for the first time that CLANs can be induced by aqueous humor. Gerometta et al (2004) Arch Ophthalmol 122; 1492-1497
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