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H. Kasai, R. Takahashi, E. Shirasawa; Collagen Gel Contraction Assay Identifies Mediators of Trabecular Meshwork Contractility . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3691.
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Purpose: Changes in the tension of the trabecular meshwork (TM) contractile affect changes in outflow rate. Smooth muscle–relaxing substances increase conventional outflow rates whereas agents that increase tension have an opposite effect leading to a rise in intraocular pressure. Three dimensional collagen gel culture is a convenient and effective to evaluate trabecular meshwork contractility. We report here on the effects of different agents that affect contractility of isolated porcine trabecular meshwork cells (pTMC) with this assay system. Methods: pTMC obtained from outgrowth of porcine TM tissues were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). pTMC embedded in type I collagen were polymerized in a 24–multiwell plate and then freed from the side of the well with a microspatula. . TMC were cultured for 48 h in the presence of different mediators (10–8 to 10–5 M) by overlaying them on the gel. At the end of this period, the gels were stained with crystal violet and their areas were calculated based on measurements of the diameter. Cell viability was determined based on reducing activity of resazulin. Results: At 10–6 and 10–5M collagen gel contraction was inhibited following exposure to either: 1) ATPase inhibitors [ethacrynic acid and thapsigargin]; 2) ion channel blockers (glibenclamide and nitrendipene); 3)HMG–CoA reductase inhibitors [pravastatin sodium or simvastatin]; 4) protein kinase inhibitors [Y27632, HA–1077, wortmannin, LY294002, roscovitine, bisindolylmaleimide, staurosporin, different tyrphostins and chelerythrine chloride] or 4) receptor agonists/antagonists [N6–cyclohexyladenosine, metrifudil, SCH23390 quinpirole]. However, thapsigargin, simvastatin, chelerythrine chloride were nonselective because they also had cytotoxic effects. On the contrary, PDGF–BB and phorbol 12 myristate elicited dose dependent ( 10–10 to 10–8 M) increases in collagen gel contraction. Conclusions: The collagen gel contraction monitoring system provides a rapid and sensitive screening method for identifying novel mediators of TMC relaxation. This technique will identify crucial determinant pathways that affect increases in conventional outflow facility through increases in TMC relaxation.
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