Objectives/Hypothesis-The avian cochlea regenerates hair cells following aminoglycoside treatment through supporting cell proliferation. Immunocytochemical labeling of BrdU, a thymidine analog, is a popular nonradioactive marker for identifying cells in the DNA Synthesis (S phase) of the cell cycle. However, it requires harsh treatments to denature double-stranded DNA for the antibody to bind BrdU. We explored a new method using EdU as a thymidine analog and a non-antibody azide/alkyne reaction between the EdU and the fluorescent probe. We propose that EdU is as effective as BrdU but without the requirement for harsh denaturation or the use of antibodies for detection.Study Design-Two week-old chicks received a single gentamicin injection followed by a single EdU injection 72h later. Cochleae were extracted 4-8h later, fixed, and processed for fluorescent detection of EdU.Methods-Cochleae were processed for detection of incorporated EdU using the Click-iT™ Imaging Kit (Invitrogen) and co-labeled with Sox2, myosin VI, or myosin VIIa antibodies. Whole-mount cochlear preparations were examined with confocal microscopy.Results-Supporting cells incorporated EdU into their newly synthesized DNA during the 4-8h following the EdU injection and were readily detected with little background signal. The intensity and quantity of cells labeled were similar to or better than that seen for BrdU. Conclusions-TheEdU method is as effective as BrdU without requiring harsh denaturation or secondary antibodies to identify proliferating cells. Thus, the non-antibody EdU system allows more flexibility by enabling co-labeling with multiple antibodies to other cellular proteins involved in regeneration.

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 initiator 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 to compare two different methods of caspase detection: caspase antibodies and CaspaTag kits. Caspase antibodies bind to the cleaved activated form of caspase-9 or caspase-3 in specific locations in fixed tissue. CaspaTag is a fluorescent inhibitor that binds to a reactive cysteine residue on the large subunit of the caspase heterodimer in unfixed tissue.To induce cochlear hair cell loss, 1-2 week-old chickens received a single injection of gentamicin (300 mg/kg). Chicks were sacrificed 24, 30, 42, 48, 72, or 96 h after injection. Cochleae were dissected and labeled for activated caspase-9 or caspase-3 using either caspase-directed antibodies or CaspaTag 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; however, caspase-9 and caspase-3 antibodies labeled only those cells currently undergoing apoptotic cell 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.

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