In our study we detected activation of three members of the caspase family, caspase-3, -8, and -9, in the OHCs of chinchilla cochleas exposed to intense noise. We also examined the release of mitochondrial cytochrome c in the noise-damaged OHCs. The significant findings of this study are as follows: (1) In apoptotic OHCs as featured by nuclear condensation or fragmentation, caspase-3 was activated, whereas in necrotic OHCs characterized by nuclear swelling, there was no caspase-3 activation. Once caspase-3 was activated, its activity remained high in apoptotic OHCs well after the apoptotic nuclei were totally disintegrated. (2) Both caspases-8 and -9 were activated after exposure to noise. Activation of caspases-8 and -9 occurred only in apoptotic OHCs in a pattern similar to that of caspase-3 activation. (3) Release of cytochrome c from mitochondria took place in both necrotic and apoptotic OHCs. Also, cytochrome c release in OHCs appeared to take place at an early stage of the cell death process. (4) Apoptotic and necrotic cells appear in approximately equal numbers and are easily distinguished using a combination of TUNEL/PI labeling methodology.
The essential role of caspase-3 in the killing process of the apoptotic cell has been recognized in a multitude of different cell lines and under various pathological conditions. It has been noted that activated caspase-3 targets the degradation of numerous intracellular and extracellular proteins that are essential for maintaining normal cell function. In a previous study (Hu et al. 2002b
), cleavage of F-actin, an important cytoskeletal protein, was found in apoptotic OHCs. F-actin cleavage was temporarily inhibited by pretreating cochleas with a caspase-3 inhibitor. In other studies, caspase-3 has been reported to be responsible for mediating DNA fragmentation by endonucleases in apoptotic cells (Cohen 1997
; Nicholson and Thornberry 1997
It has been noted that the time required for activation of caspase-3 is associated with specific experimental conditions, such as the nature of apoptotic stimuli and cell line used. The time required for caspase activation induced in the brain either by ischemia (Han et al. 2000
; Sasaki et al. 2000
; Velier et al. 1999
) or chemical stimuli (Kondratyev and Gale 2000
; Asakura et al. 1999
; Hayami et al. 1999
) ranges from several hours to one day after exposure to an apoptotic stimulus. In our study, we found that activation of caspase-3 took place either shortly after noise exposure or possibly during noise exposure. Considering that (1) activation of caspase-3 was found in the processed cochlea immediately after noise exposure and that (2) the total length of time
(2.5 h) required for completing the experimental manipulation, it is likely that activation of caspase-3 occurs within 2.5 h after noise exposure is initiated. This estimated time frame is based on the length of time required for complete processing of cochleas, which includes the noise exposure, the surgical procedure for the perfusion of caspase-3 probe, and the subsequent incubation period. The time required for caspase activation in apoptotic OHCs is unusually brief and may be a consequence of the high-intensity noise utilized. A low-intensity noise may require a longer time period for caspase-3 activation and requires further investigation.
Examination of OHC nuclei in this study, as well as in a previous study (Hu et al. 2002a
), revealed a rapid development of OHC pathologies during high level noise exposure. Immediately after exposure to noise, apoptotic nuclei appeared while others were missing in damaged sections of the cochlea, particularly at the damaged center. Also, at this time point, F-actin associated with the cuticular plates of apoptotic OHCs show sections of discontinuity that are indicative of breakdown of F-actin (Hu et al. 2002b
). These early changes in F-actin breakdown coincided with rapid activation of caspase-3 and its inhibition prevented F-actin cleavage. These results clearly indicate the involvement of caspase-3 in OHC apoptosis induced by exposure to intense noise. Activated caspase-3 initiates the hydrolytic breakdown of intracellular and extracellular proteins and, consequently, the complete degradation of the apoptotic cell takes place in a relatively short time.
In addition to its rapid activation, caspase-3 activity persists in dying OHCs well after nuclei are degraded. However, the ultimate fate of apoptotic OHCs is not as yet clear. Apoptotic bodies are generally formed, which consist of nuclear fragments and intact organelles enclosed by a plasma membrane fragment. After their formation, these apoptotic bodies are subsequently engulfed by neighboring cells or macrophages and cleared from the lesion (Nishikawa et al. 1998
; Rubartelli et al. 1997
). Fredelius and Rask–Andersen (1990)
reported that macrophages are present in the organ of Corti and survive for five days after noise exposure. Our study did not identify any obvious presence of macrophages within the organ of Corti or any sign of engulfment of apoptotic bodies by supporting cells after noise exposure. A more careful detection of macrophages, such as antibody recognition of surface antigens, is required to confirm their presence in the cochlea and to further characterize the mechanism(s) by which apoptotic bodies are removed from the cochlea. It is possible that caspase-3 remains activated until the cell is either sufficiently degraded or the remnants removed by scavenger cells.
Activation of caspase-3 is a downstream event in the apoptotic cascade, which can be triggered by a variety of extracellular or intracellular signaling pathways. To date, two pathways, the cell death receptor-mediated pathway and the mitochondrial pathway, have been well characterized in many cell lines to converge on caspase-3 activation. In the receptor-mediated pathway, ligands such as Fas or tumor necrosis factor α bind to its receptor, causing receptor aggregation and recruitment of death adapter molecules on the cytoplasmic side of the membrane (Ashkenazi and Dixit 1998
). Procaspase-8 is recruited to the complex and cleaved to form activated caspase-8. Activated caspase-8, in turn, cleaves procaspase-3 and activates it. In the mitochondrial pathway, a proapoptotic member of the bcl-2 family, such as bax or bid, associates with the mitochondrial pore complex and directs the dissociation and the eventual release of cytochrome c to the cytosol where it can associate with another key factor, Apaf-1. Apaf-1 binds cytochrome c, dATP, or ATP and forms a large multimeric complex, which includes caspase-9. Caspase-9 is activated upon binding to Apaf-1, whereby this complex processes procaspase-3 to its active form.
In noise-induced apoptosis, information is not as yet available as to which pathways are responsible for the activation of caspase-3. We therefore examined the activation of both caspases-8 and -9 in response to noise. We found that both caspase-8 and caspase-9 were activated in apoptotic OHCs following noise exposure in a pattern similar to caspase-3 activation. Neither of these caspases was found in an activated state in necrotic OHCs. A more detailed analysis of the temporal sequence for activation of caspases-8 and -9 using a concurrent fluorescence labeling procedure in noise-damaged cochleas was not possible due to the similarity in spectra of the fluorescent probes used in this study. Attempts to reduce the noise intensity yielded an inconsistent apoptotic response that precluded analysis. Therefore, it is possible that individual OHCs underwent apoptosis either through the caspase-8 or caspase-9 pathway alone. However, this possibility appears unlikely because probes for both caspases-8 and -9 fluoresce in virtually all of the OHCs with condensed or fragmented nuclei. Dual staining of caspases-8 and -9 using different fluorophores could yield a more definitive outcome as to their activation sequence.
Understanding the relative contribution of caspases-8 and -9 and their activation sequence are important issues that should be pursued. For instance, caspase-8 activation could provide information regarding a contribution from ischemia/reperfusion and subsequent signaling through cell death receptor pathways, whereas caspase-9 could indicate a contribution of mitochondrial overstimulation. Unfortunately, additional experiments yielded little useful information. Use of specific, nonfluorescent inhibitors for either caspase-8 or caspase-9 did not significantly reduce caspase-3 activation, and a pan-caspase inhibitor only slowed the apoptotic process but did not eliminate it (data not shown). One possible explanation for this data is that not all activated caspases have been identified in response to noise stimulation. We are currently testing whether, in fact, additional caspases are being activated by noise.
Translocation of cytochrome c from the mitochondria has been reported to take place in many cell systems during apoptosis and its release is followed by the activation of caspase-9, which, in turn, activates caspase-3 (Cai et al. 1998
). The mechanism for the translocation of cytochrome c to the cytosol is not completely understood. Whether mitochondrial swelling and consequent disruption of the outer membrane permits the release of cytochrome c, or whether bcl-2 family members regulate cytochrome c release through their capacity to form membrane channels, is currently being debated. What is clear, however, is that release of cytochrome c is closely associated with apoptosis (Zhivotovsky et al. 1998
). In contrast to these reports, our observation is that the release of cytochrome c was associated with both apoptotic and necrotic OHCs. The biological significance of this finding is not yet clear. Furthermore, release of cytochrome c was also noted in OHCs that demonstrated no clear indication of either necrotic or apoptotic nuclear morphology. This observation is in agreement with a previous report indicating that, in some cells, cytochrome c release is an early event that precedes caspase activation and that caspase inhibition prevents ultrastructural changes within the cell (Dinsdale et al. 1999
). An interesting possibility is that release of cytochrome c in hair cells may be a common early step that triggers cell death processing. Several factors are known to contribute to the particular cell death pathway selected; these include the level of caspases activated, the energy available in the form of ATP (Leist et al. 1997
), and the nature and severity of the insult (Bonfoco et al. 1995
Evidence is accumulating that inhibition of caspase-dependent apoptosis in many cases leads to a necrotic-like cell death (Kitanaka and Kuchino 1999
). One recent study shows that the apoptotic pathway is associated with massive release of activated caspases into the media, whereas in necrotic cells only inactive procaspases were detectable (Denecker et al. 2001
). In the cochlea, apoptosis and necrosis appear to be intertwined in their own unique fashion in a process that is only now beginning to be uncovered. What is clear is that caspases are indispensable as initiators and effectors of the apoptotic cell death program, whereas cytochrome c release is associated with both necrosis and apoptosis. To this end, we have incorporated the use of multiple markers which permits the facile discrimination between apoptotic and necrotic cells within the cochlea. The morphological changes, including cytoplasmic swelling and plasma membrane permeabilization as identified by the incorporation of PI, can be used to clearly distinguish necrotic cells, whereas apoptotic cells are readily identifiable by the formation of apoptotic bodies in combination with either DNA degradation or the release of activated caspases. These differences will serve as important markers in the stepwise characterization of the hair cell death responses to noise.