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R. Linden, C.A. Guimarães, M. Benchimol, G. Amarante-Mendes; Alternative Programs of Cell Death in Retinal Tissue . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2834.
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Purpose: To examine the morphology and mechanisms of cell death induced by inhibition of protein synthesis in the developing retina, with emphasis on the relationship between apoptosis and autophagy. Methods: Explants of the retina of neonatal rats were maintained in culture in either control medium or treated with the inhibitor of protein synthesis anisomycin. Ultrathin sections were examined by transmission electron microscopy. Frozen sections were labeled with the TUNEL technique, or stained with antibodies for activated caspases-3 or -9. Cell death was quantified in explants subject to treatment with the autophagy inhibitor 3-methyl adenine, the inhibitor of mitochondrial permeability transition bongkrekic acid, or each of several caspase inhibitor peptides. Results: Electron microscopy revealed both apoptotic and autophagic profiles in the neuroblastic layer of retinae treated with anisomycin. The TUNEL technique and immunohistochemistry for the activated form of either caspase-3 or -9 labeled roughly half of the degenerating profiles. Inhibitors of autophagy, caspase-9 or caspase-3 prevented TUNEL-positive cell death, while inhibition of caspase-6 blocked TUNEL-negative cell death. Either treatment with bongkrekic acid or simultaneous inhibition of both caspase-9 and -6 prevented cell death almost completely. However, cell death dependent on autophagy/caspase-9 still occurred under inhibition of both caspase-3 and -6. Conclusions: Inhibition of protein synthesis induces two distinct pathways of cell death in the retina, both dependent on mitochondrial commitment and caspase activation. However, caspase-9 and caspase-6 are activated in separate cascades. The activation of caspase-9 is preceeded by autophagy and leads to caspase-3 activation and DNA fragmentation, while caspase-6 leads to TUNEL-negative cell death. A third program of cell death may be engaged when both caspases-3 and -6 are inhibited.
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