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M. Moosajee, C. Y. Gregory-Evans, K. Gregory-Evans; The Anti-Apoptotic Action of Curcumin and zVAD-fmk Provides a Potential Treatment for Ocular Coloboma in the lamb1 Zebrafish Model. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4810.
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© ARVO (1962-2015); The Authors (2016-present)
Ocular coloboma arises from incomplete fusion of the optic fissure between 5-7 weeks post conception in humans and 36-48 hours post fertilisation (hpf) in zebrafish. The aetiology of this congenital eye anomaly shows extensive genetic heterogeneity, and there is no effective treatment available. Apoptosis plays a transient role in normal optic fissure morphogenesis, however, deleterious levels have been detected at the site of the unfused optic fissure in a number of animal models of ocular coloboma, including the lamb1-/- zebrafish. This study aims to identify whether drugs targeting apoptosis can provide a safe, effective and mutation-independent treatment for ocular coloboma, and improve understanding of the cellular mechanisms involved in optic fissure morphogenesis.
Mutant lamb1-/- embryos were treated with 5 µM curcumin (diferuloylmethane, a caspase-3 inhibitor, derived from Curcuma longa), 300 µM zVAD-fmk (fluoromethylketone molecule, a pan-caspase inhibitor) and 300 µM zFA-fmk (negative control, cathepsin B inhibitor) from 10 hpf; ocular morphology was examined by light microscopy at 48 hpf and day 6. The lamb1-/- mutation causes lethality by day 5; survival times were determined for treated and untreated larvae (n=50 per group). Apoptosis at the site of the optic fissure was detected by TUNEL assay.
Curcumin and zVAD-fmk-treated lamb1-/- mutants displayed a significantly milder coloboma phenotype associated with negligible TUNEL-positive staining at the site of the persistent optic fissure, compared to zFA-fmk and untreated embryos at 48 hpf and day 6. Mean survival of lamb1-/- mutants treated with both drugs resulted in a 1.4 fold increase in survival (average 7.1 days, p>0.0001 using the Mann-Whitney Test).
Both anti-apoptotic agents greatly reduced the size of the colobomatous defect by minimising cell death. This suggests optic fissure remodelling requires apoptosis, but is not wholly dependent on this mechanism. Supplemental curcumin may provide a safe prenatal treatment which limits the severity of ocular coloboma and has applications for use in other ocular developmental anomalies displaying pathological levels of apoptosis.
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