Comparison of activated caspase detection methods in the gentamicin-treated chick cochlea

Aminoglycoside antibiotics induce caspase-dependent apoptotic death in cochlear hair cells. Apoptosis, a regulated form of cell death, can be induced by many stressors, which activate signaling pathways that result in the controlled dismantling of the affected cell. The caspase family of proteases is activated in the apoptotic signaling pathway and is responsible for cellular destruction. The intiator caspase-9 and the effector caspase-3 are both activated in chick cochlear hair cells following aminoglycoside exposure. We have analyzed caspase activation in the avian cochlea during gentamicin-induced hair cell death of compare two different methods of caspase detection: caspase antibodies and CaspaTag kits. Caspase anti-bodies bind to the cleaved activated form of caspase-9 or caspase-3 in specific locations in fixed tissue. CaspaTag is fluorescent inhibitor that binds to a reactive cysteine residue on the large subunit of the caspase heterodimer in in fixed tissue. To induce cochlear hair cell loss, 1-2 week-old chickens received a single injection of gentamicin (300 mg/kg.) Chicks were caspase-9 or caspase-3 using either caspase-directed anitbodies or CapaTag kits. Ears were co-labeled with either phalloidin or myosin VI to visualize hair cells and to determine the progression of cochlear damage. The timing of caspase activation was similar for both assays; how-ever, caspase-9 and caspase-3 antibodies labeled only those cells currently undergoing apoptotic cells death. Conversely, CaspaTag-labeled all the cells that have undergone apoptotic cell death and ejection from the sensory epithelium, in addition to those that are currently in the cell death process. This makes CaspaTag ideal for showing an overall pattern or level of cell death over a period of time, while caspase antibodies provide a snapshot of cell death at a specific time point. (C) 2008 Elesvier B.V. All rights reserved.