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The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration
Published on: November 26, 2015
Comparison of activated caspase detection methods in the gentamicin-treated chick cochlea
Christina L Kaiser1, Brittany J Chapman, Jessica L Guidi
1Laboratory for Cellular and Molecular Hearing Research, Department of Otolaryngology, Evans 637, Boston University Medical Center, 715 Albany Street, Boston, MA 02118, USA. ckaiser@bu.edu <ckaiser@bu.edu>
Abstract:
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.
Insights
Aminoglycoside antibiotics cause hair cell death via apoptosis. This study compares caspase antibodies and CaspaTag kits for detecting apoptosis in cochlear hair cells, finding CaspaTag better for overall cell death patterns.
Area of Science:
- Ototoxicity and molecular mechanisms of cell death.
- Cellular biology and signaling pathways.
Background:
- Aminoglycoside antibiotics are known to induce apoptosis, a programmed cell death, in cochlear hair cells.
- Caspases are key proteases activated during apoptosis, leading to cellular dismantling.
Purpose of the Study:
- To compare the efficacy of two methods, caspase antibodies and CaspaTag kits, for detecting caspase activation in gentamicin-induced cochlear hair cell death.
- To analyze the timing and pattern of apoptosis in avian cochlear hair cells following aminoglycoside exposure.
Main Methods:
- Gentamicin was administered to young chickens to induce cochlear hair cell death.
- Cochleae were analyzed at various time points post-injection using either caspase-directed antibodies or CaspaTag kits to detect activated caspase-9 and caspase-3.
- Hair cells were visualized using phalloidin or myosin VI to assess cochlear damage progression.
Main Results:
- Both caspase antibodies and CaspaTag kits detected caspase activation, with similar timing.
- Caspase antibodies identified cells actively undergoing apoptosis, while CaspaTag labeled all cells that had undergone or were undergoing apoptosis.
- CaspaTag provided a comprehensive overview of cell death extent over time, whereas antibodies offered a specific temporal snapshot.
Conclusions:
- CaspaTag kits are more suitable for evaluating the overall pattern and extent of aminoglycoside-induced cochlear hair cell death over time.
- Caspase antibodies are useful for pinpointing specific cells in the process of apoptotic death at a given moment.
