Apoptosis is responsible for cochlear cell death induced by noise. Here, we show that transgenic (TG) mice that overexpress X-linked inhibitor of apoptosis protein (XIAP) under control of the ubiquitin promoter display reduced hearing loss and cochlear damage induced by acoustic overstimulation (125 dB sound pressure level, 6 h) compared with wild-type (WT) littermates. Hearing status was evaluated using the auditory brainstem response (ABR), whereas cochlear damage was assessed by counts of surviving hair cells (HCs) and spiral ganglion neurons (SGNs) as well as their fibers to HCs. Significantly smaller threshold shifts were found for TG mice than WT littermates. Correspondingly, the TG mice also showed a reduced loss of HCs, SGNs and their fibers to HCs. HC loss was limited to the basal end of the cochlea that detects high frequency sound. In contrast, the ABRs demonstrated a loss of hearing sensitivity across the entire frequency range tested (2–32 kHz) indicating that the hearing loss could not be fully attributed to HC loss alone. The TG mice displayed superior hearing sensitivity over this whole range, suggesting that XIAP overexpression reduces noise-induced hearing loss not only by protecting HCs but also other components of the cochlea.
Exposure to hazardous noise is the primary cause of hearing impairment in North America.1 Noise-induced hearing loss (NIHL) is thought to result primarily from damage to the organ of Corti (OC) in which hair cells (HCs), particularly outer hair cells (OHCs), are considered the most vulnerable.2 Although OHC loss is a contributing factor to NIHL, hearing deficits are not closely correlated with the number of missing OHCs, suggesting that the death of other cell types in the cochlea has an important role in the development of NIHL.3, 4, 5 For example, loss of spiral ganglion neurons (SGNs)6 and cell death in structures outside the OC such as the stria vascularis may also contribute to NIHL.7, 8, 9, 10, 11 Both necrosis and apoptosis have been implicated in NIHL.12, 13, 14 Their relative contribution to noise-induced cochlear cell death changes with the types of acoustic exposures and the location along the cochleae relative to the center region where damage is centered on.12, 13, 14, 15 Nevertheless, apoptosis is thought to have a significant role in noise-induced damage of the cochlea.14, 16, 17
Apoptosis may be executed by a family of cysteine proteases called caspases.18, 19, 20 To date, 14 caspases have been identified that are broadly divided into two groups, initiator (caspase-6, -8, -9 and -10) and executioner (caspase-2, -3 and -7).18, 21, 22 In NIHL, apoptosis resulting from activation of the extrinsic pathway (caspase-8) or the intrinsic pathway (caspase-9) has been observed.12, 13, 15, 16 These two pathways converge on caspase-3, the final executioner of apoptosis. Both the intrinsic and extrinsic pathways are opposed by members of the inhibitor of apoptosis protein (IAPs) family.23, 24, 25 The IAP family consists of eight members that have at least one baculoviral inhibitor of apoptosis repeat domain. The IAPs play a central role in cell survival such that a reduction of the apoptotic threshold occurs upon their downregulation.26, 27 Whereas IAP overexpression increases resistance to a variety of triggers of apoptotic cell death.28, 29, 30 Among the IAP family members, X-linked inhibitor of apoptosis (XIAP) is considered to be the most potent because of its ability to strongly suppress caspase-3 activity.31, 32 In the case of in vivo models for stroke and Parkinson's disease, XIAP overexpression not only renders neurons more resistant to cell death but preserves normal function.33, 34, 35
The C57BL/6J mouse strain is known for its rapid development of hearing loss with aging.36, 37, 38, 39 Using C57BL/6J mice that overexpress human XIAP under control of the ubiquitin promoter (ubXIAP), we have found that in comparison with wild-type (WT) littermates, ubXIAP mice showed a delay in the development of presbycusis, which was also associated with a reduction in cochlear HC loss.40 In this study, we have used these transgenic (TG) mice to determine the effects of XIAP overexpression on NIHL and associated cochlear damage.
Auditory brainstem response (ABR) threshold shifts after acoustic overstimulation
Figure 1 shows the ABR threshold shifts 1 and 4 weeks after exposure to acoustic overstimulation of 125 dB sound pressure level (SPL) for 6 h. Although the acoustic overstimulation occurred at two frequencies (1 and 6 kHz), the threshold shifts appeared to be greater at middle to high frequencies (4 kHz and above) than at the lower frequency (2 kHz). The baseline recording before acoustic overstimulation shows the ABR thresholds are equivalent between the two groups (Table 1). There was a rapid initial partial recovery in ABR thresholds shortly after the acoustic overstimulation (data not shown). However, no further recovery was found after 1 week so that the two sensitivity curves for each group obtained at 1 and 4 weeks after the acoustic overstimulation were virtually identical to those obtained at 1 week. Most importantly, the threshold shifts were much less (by 10 to 25 dB) in the TG than in the WT group (Figure 1). An analysis of covariance (fixed factor: genotype; covariate: frequency) identified a significant effect of genotype (F1,147=19.468, P<0.001). Post-hoc tests (t-test with Bonferroni correction) showed that significance was found between groups at all frequencies at both time points (1 and 4 weeks). These results indicate that TG mice engineered to express higher than normal levels of XIAP in the cochlea were protected against NIHL.
Cytocochleograms were successfully established for 19 cochleae from TG mice group and 16 cochleae from WT controls. Figure 2 shows the average IHC (left) and OHC (right) loss resulting from the acoustic overstimulation as a function of percent distance from the cochlear apex. Unlike the widespread functional hearing loss, which encompassed the low-frequency transducing regions of the cochlea, the actual anatomical losses of both IHCs and OHCs in both groups were limited to the basal end of the cochlea (within the region for sound frequencies above 10 kHz).
A significant amount of HC loss was seen in both groups, although HC loss was much less in TG than WT mice. This indicates that compared with WT mice, TG mice that overexpress XIAP displayed reduced HC loss after acoustic overstimulation. The averaged total HC loss per cochlea (IHC+OHC loss) in the WT group was 501.76±247.68 (mean±s.e.m.) per ear, whereas the corresponding number for the TG mice was 240.37±207.80 per ear. The data of both ears from each animal were further averaged to generate the number of HCs for each individual in which two cochleae were used for HC evaluation. The difference was further compared across groups and was found to be statistically significant (t28=3.632, P=<0.001). The loss of both IHCs and OHCs show a similar trend. However, the percentage of IHC loss is much less prominent than that seen in the OHCs in both WT and TG groups, and was more restricted to the higher frequency region.
Figure 3 shows representative images of surface preparations of the cochlea in which sensory HCs from both WT and TG mice exposed to acoustic overstimulation are visible. These eight low-magnification images represent four distinct descending levels from the apical (top four panels) to basal (bottom four panels) turns of the cochlea. These four segments from each cochlea cover more than 90% of the entire cochlea. In the representative WT mouse, HC loss (mostly the OHCs) was restricted to the extreme basal end of the cochlea that encompassed the hooked region (bottom left panel). HC loss was greater in WT (bottom left panel) than TG mice (bottom right panel).
Damage to SGNs and their fibers
The cochlear lesion induced by the acoustic trauma was further evaluated in terms of the damage to SGN cell bodies and their dendrites to IHCs (nerve fibers). Figure 4 shows typical images of cochlear cross-sections stained with Toluidine blue. SGN cell bodies were counted at four locations as indicated in Figure 4a (1–4). The insert shows a magnified image of location 1 where SGN cell body counts were performed. The a–a line in Figure 4a indicates the location and the orientation of the cross-section revealing habenular perforatae in which the number of auditory nerve fibers (SGN dendrites) were counted.
The first finding from this cross-section analysis was that, acoustic trauma produced a reduction of the number of dendrites across a larger region of the cochlea compared with HC loss seen in the cytocochleogram. In Figure 4b, the arrow indicates habenular perforatae in the apical turn where an obvious loss of dendrites is seen. In this region, however, the threshold shift in ABR was less and no sensory HC loss was found. Figure 5 demonstrates exemplary images of the habenular perforatae from the basal turn of a cochlea of a no-acoustic overstimulation control at low magnification (top panel) and high magnification (bottom panel). Individual dendrites can be seen in the bottom panel of Figure 5, which were counted to assess fiber loss. Figure 6 shows the images taken at the levels of the basal (upper panel) and apical turns (lower panel) of a cochlea damaged by the acoustic overstimulation. In the middle panels of Figure 6, a massive loss of dendrites was evident in both turns (left middle panel, basal turn; right middle panel, apical turn). Therefore, unlike the loss of HCs, which was restricted to a small region of the cochlea (Figure 3, lower left panel), the acoustic overstimulation produced a widespread loss of SGN dendrites in the cochlea (Figure 6).
To quantitatively evaluate the severity of the auditory nerve lesion, the number of SGN dendrites were counted in 10 habenular perforatae, respectively, at the apical and basal turns for each cochlea. The total number of dendritic fibers counted in the 20 habenular perforatae were 2160±48 (n=10, control), 1300±113 (n=10, WT acoustic overstimulation) and 1586±53 (n=10, TG acoustic overstimulation) for the no-acoustic overstimulation control, WT and TG groups, respectively. Although fiber numbers for both acoustically-damaged groups (WT and TG) were significantly lower than the non-acoustic overstimulation control, TG mice had significantly more dendritic fibers than WT mice (t18=2.434, P=0.026). Hence, XIAP overexpression also protected against the loss of dendritic fibers arising from SGNs.
Similar to the loss of SGN dendrites there was a massive loss of SGN cell bodies following the acoustic overstimulation. Figure 7 shows images of SGN cell bodies in a cochlear cross-section through the Rosenthal canal of a no-acoustic overstimulation control (left panels), and WT animal exposed to acoustic overstimulation (right panels). The upper panels show images from the apical turn and the lower panels correspond to the basal turn of the cochlea. In each cochlea, SGN cell bodies were counted at four locations: twice at both the basal and apical turn. At each of these levels the numbers of SGN cell bodies were averaged from two sections that were separated by over 100 μm in distance. The total of SGN cell body counts from the four levels was used as the index of SGN cell body density for each cochlea. The averaged counts for each ear were 308±11 (n=10), 222±7 (n=10) and 261±4 (n=10) for the control, WT and TG groups, respectively. Although the acoustic overstimulation reduced the number of SGN cell bodies in both WT and TG groups relative to no acoustic overstimulation controls, the reduction in SGN cell bodies for the TG group was significantly smaller (t18=2.44, P=0.02) than that of the WT group. Consequently, elevated XIAP levels in TG mice also reduced the loss of SGN cell bodies compared with WT littermates exposed to acoustic overstimulation.
Western blotting for XIAP
Both ub- and endo-XIAPs were analyzed in a semi-quantitative manner by densitometry using β-actin, a housekeeping protein, as a loading control. The levels of both XIAPs were normalized according to the levels of β-actin.
The level of ubiquitous X-linked Inhibitor of Apoptosis Protein (ub-XIAP) was found to be unchanged by acoustic overstimulation (0.654±0.039 in control mice, n=5 (10 ears from five mice) versus 0.634±0.021, n=15 (30 ears from 15 mice) in the group of acoustic overstimulation, t17=0.739, P>0.05). The level of endo-XIAP in the control mice (without acoustic overstimulation) was 0.564±0.64, and 0.441±0.043 for the TG and WT group, respectively, with no significant difference (P>0.05). The endo-XIAP levels were higher in the corresponding groups exposed to acoustic overstimulation (0.718±0.041 for the TG group and 0.808±0.053 for the WT group, n=15 for each). Figure 8 shows the ratio of endo-XIAP to β-actin in both the control and groups exposed to acoustic overstimulation. one-way analysis of variance was performed on two factors: genotype and acoustic overstimulation, after the conversion of ratio values to their square roots. The effect of acoustic overstimulation was found to be significant (F1,79=23.37, P<0.001), whereas the effect of genotype was not significant (F1,79=0.307, P>0.05). However, there was a significant interaction between the two factors (F1,78=4.9, P=0.029), which is evident by a larger noise-induced increase of endo-XIAP in the WT group (from 0.441±0.043 in the control to 0.808±0.053 in the subgroups with acoustic overstimulation) than in the TG group (from 0.564±0.64 in the control to 0.718±0.041 in the subgroups with acoustic overstimulation).
The key finding of this study is that mice with overexpressed XIAP displayed reduced NIHL compared with their WT littermates. WT mice developed greater threshold shifts after acoustic trauma than TG mice throughout the frequency range tested. Correspondingly, the loss of sensory HCs, as well as SGNs, and their dendrites to the HCs were significantly less in the TG than WT group. These results suggest that XIAP overexpression reduced NIHL, at least in part, by decreasing noise-induced damage to the cochlea. In addition to the protective effect of XIAP overexpression against acoustic overstimulation, it is also possible that the inner ears of TG mice were healthier before the acoustic trauma because TG mice showed a delayed development of presbycusis, as reported in our previous study.40 However, this potential confounding factor can largely be ruled out because the control recordings of ABR do not show any difference in hearing status across groups (Table 1) and there is no difference in HC counts in TG mice and WT mice at this age when the cochleogram normative values were established in our lab (data not reported).
Cumulative evidence suggests that the acoustic overstimulation can cause cell death in the cochlea by several different but overlapped pathways.12 Noise drives excessive mitochondrial activity, resulting in the formation of reactive oxygen species that contribute to apoptotic death in the OC.41 The JNK pathway, known to be redox regulated and pro-apoptotic, has been implicated in inner ear cell death produced by ototoxins (aminoglycosides) and excessive noise.42, 43 In recent years, the mechanical effect of noise on the point of contact of HCs to the surrounding cells and the extracellular matrix has been recognized as a trigger for a specific apoptotic cell death called anoikis.14, 44, 45, 46, 47 However, this type of cell death may also be due to oxidase activation. XIAP is thought to inhibit both JNK signaling and reactive oxygen species production by activating the NF-kappaB pathway.48 Consequently, XIAP may reduce NIHL by opposing both caspase-dependent and -independent cell death pathways. These multifaceted activities make XIAP an attractive therapeutic candidate for the prevention of NIHL by gene therapy.
Various intervention strategies have been evaluated to protect cochleae from noise-induced damage. Many previous studies have used antioxidant methods to suppress reactive oxygen species, which may be overproduced by noise exposure.49, 50, 51, 52, 53, 54, 55 Although variation exists in the degree of protection offered by these agents, significant protection has been identified both functionally as reduced threshold shift and morphologically as decreased HC loss in these antioxidant therapeutic studies. Consistent with a role for the JNK pathway in noise-induced cochlear cell death, inhibitors suppressing this stress kinase pathway have also been shown to be protective.56, 57
Tearing and stressing of the cell junctions between HCs have been observed in the noise-exposed cochlea.46, 51 Decreased cell attachment or cell stress can activate members of the Src family of tyrosine kinases resulting in cell death by a process called anoikis. Experimentation using chemicals that inhibit Src kinases showed that anoikis also contributes to NIHL.58, 59 Although quantitative comparison of the degree of protection produced by different protective strategies for NIHL is impossible because of differences in species, noise-exposure regimen and the morphological methods, the degree of reduced HC loss and the threshold shift observed in this study are comparable, if not superior to that reported for alternative treatments.
In most of these previous studies reviewed above, exogenous protective agent(s) was/were applied systematically or by cochlear infusion. As these agents will be removed by metabolism, the protection may last for only a short period of time. This study suggests an approach by manipulation of one or more genes regulating cell death. Such manipulation can be performed through cochlear gene therapy, which will provide long-term protection. A study is underway in our laboratory to explore the technology necessary for this application.
Nevertheless, none of these treatments completely protect against NIHL, suggesting that noise causes cochlear cell death by multiple pathways, which cannot be prevented completely by blocking a single pathway. Although XIAP overexpression reduced NIHL in this study, protection was not complete for which there are several potential reasons. First of all, hearing loss induced by noise results in part from necrotic damage that cannot be prevented by XIAP. Furthermore, lesions around HCs can cause malfunction without resulting in cell death. Discrepancies between the actual amount and distribution of HC (especially OHC) death and the threshold shifts have been well reported.3, 60, 61, 62, 63 Non-lethal damage to HCs and accessory structures in the OC have long been considered to have essential roles in NIHL. One of the most likely loci for this is the stereocilia and tip links.3, 4, 63, 64 Another possibility is damage to the lateral walls of the OHCs, especially structures related to the function of prestin,5, 61, 62, 65, 66 the motor protein critical for the function of OHCs as a mechanical amplifier.67, 68, 69, 70
The acoustic overstimulation used in this experiment was intense and produced a widespread threshold shift, although larger threshold shifts were seen above 4 kHz. At the lowest frequency tested (2 kHz), the threshold shifts in both groups were small. Such deterioration in hearing function across a large frequency area is more likely because of the damage to structures involved in cochlear transduction and mechanical amplification. Such damage is unlikely to occur by apoptosis and is therefore beyond the scope of protection exerted by XIAP overexpression.
Data in several recent reports and in our present study show widespread noise-induced lesions in the auditory N-terminal and SGN loss in mice.4, 6, 63 This is interesting because the damage to the dendrites of SGNs, and even the death of SGNs, were seen in regions of the cochlea where there was no significant HC loss and limited or no functional hearing loss. However, more experiments are needed to see if this lesion can occur in other species that possess bigger cochleae and therefore have a longer distance between the IHC-SGN synapses and the soma of SGNs. In this study, a similar loss in auditory nerve fibers was found 1 month after the acoustic overstimulation. However, the partial loss of SGNs may not be responsible for the threshold shift. Measurements taken at this time identified a smaller loss of SGNs than of nerve fibers. More SGN loss is expected if sampling at longer intervals after the acoustic overstimulation, according to previously published data.6
A second possible reason for the limited protection in this TG model is because of the limited elevation of the XIAP level and the potential interaction between the ub-XIAP and the endo-XIAP. The exogenous xiap gene promoted by ubiquitin resulted in a doubling of total XIAP level in the control cochleae. As apoptosis is an important physiological mechanism for controlling early development, it would be a concern to increase the XIAP too highly. Otherwise, animals may not go through normal development. In addition, the level of the ub-XIAP was constant because of the lack of regulating zones in the transgene and therefore it did not respond to the stress of acoustic overstimulation. Furthermore, the acoustic overstimulation-induced elevation of endo-XIAP level is relatively small in the TG group, indicating potentially a negative feedback from the ub-XIAP to the regulating mechanism for the endo-XIAP.
It is also possible, because of the high levels of the acoustic overstimulation, that the internal protective mechanisms mediated by XIAP are not activated promptly enough to protect the cochlear cells. Therefore, a constant high level of exogenous XIAP may be required for an ideal protection. This can be achieved by local gene transfection, thus, avoiding the potential impact on development and other side effects (such as tumor growth) of TG manipulation at the genome level.
In summary, this study demonstrated C57BL/6J mice with overexpressed XIAP displayed partial resistance to NIHL and displayed a reduced cochlear cell death. This protection resulted in smaller threshold shifts and a reduction in the loss of IHCs, OHCs, SGNs and auditory nerve fibers in TG mice. The shifts in auditory thresholds were present across the entire frequency range; however, HC damage was restricted to the basal end of the cochlea demonstrating an indirect relationship between HC loss and the functional measures.
Materials and methods
Subjects and experiment overview
TG founders were generated as previously described.71 Briefly, a linearized plasmid construct consisting of the Ubiquitin C promoter, six repeats of the 9E10 myc epitope tag fused to the amino terminus of the human XIAP coding region, and a polyadenylation signal from stria vascularis-40 was microinjected into the male pronucleus of C57BL/6J X C3 H F1 zygotes. C57BL/6J X C3 H F1 offsprings were backcrossed over 15 generations against the WT C57BL/6J mice to obtain ub-XIAP TG animals on a pure C57BL/6J genetic background. To obtain WT littermates as controls, ub-XIAP animals were crossed with WT C57BL/6J mice. TG status within the colony was determined by PCR targeting the 6-myc tag. All TG mice and their WT littermates used in this experiment were bred in the Facility for Animal Care, Dalhousie University, Halifax, NS, Canada. The impact of acoustic trauma on hearing function and cochlear morphology was examined in two groups of mice, one comprised of 15 ubXIAP (TG) animals and the other 15 WT littermates. These mice were recruited into the experiment at the age of 1–1.5 months. Frequency-specific ABR were recorded as an index of hearing status before and at different time points up to 1 month after the acoustic overstimulation. Those animals with abnormal hearing, verified by a baseline test, were excluded. After the final ABR test, all mice were killed under deep anesthesia and their cochleae were harvested. From the first five mice in each group, both ears were used for cytocochleograms in surface preparation for HC loss. However, any two cochleae that were taken from one animal were considered as one sample point by averaging their HC count values in the statistical analyses. In the remaining 10 mice in each group, one ear from each mouse was used for cytocochleograms and the other for evaluating the damage to SGNs and nerve fibers by cross-sectioning. In addition, six cochleae from six mice experiencing no acoustic trauma were used to establish control norms for the axon fibers and SGN counts. To evaluate the impact of acoustic overstimulation on the level of XIAPs (both endogenous and that from the transgene), an additional 40 animals (20 WT and 20 TG) were used for western blot analysis. In each genotype, 10 cochleae from five mice were used as no-noise controls and the other 30 cochleae from 15 mice were harvested 24 h after exposure to the acoustic overstimulation.
For ABR recordings, mice were anaesthetized with Ketamine+Xylazine (80–100 mg kg−1 +10 mg kg−1, respectively, i.p.). An additional one-fourth of the initial dose was administered as needed to maintain anesthesia. The mice were placed on a thermostatic heating pad to ensure the body temperature remained at 38.5 °C during the procedure. Tucker–Davis hardware and BioSig software (Tucker–Davis Technology system III, Alachua, FL, USA) were used for the signal generation and acquisition of ABR in response to tone bursts of 2–32 kHz in octave steps. The tone burst, with a duration of 10 ms and rise/fall of 1 ms, was delivered through a broadband electrostatic speaker (ES1 from TDT) which was placed 10 cm in front of the animal's head in a sound-proof booth. At each frequency, the signal was presented from 90 dB SPL down to 10 dB SPL in 5–10-dB steps. The sound level was calibrated using a 1/4-inch B&K (Skodsborgvej, Denmark) condenser microphone (Model 4939), which was placed at the position that would be occupied by the head of the animal. The output of the microphone was examined using SigCal software from TDT.
Subdermal electrodes were used for ABR recording, with the active recording electrode on the vertex of the skull and the reference, and ground behind each ear. The responses were band-pass filtered between 100–3000 Hz, amplified and averaged over 1000 times with a repetition rate of 21.1 s−1. The threshold was judged as the lowest SPL at which a repeatable response was visible. If no waveform was identified at the highest presentation level (90 dB SPL) for a particular frequency, the threshold was then documented as 100 dB SPL.
Mice were exposed to the acoustic overstimulation for 6 h in a sound proof booth, unanesthetized and unrestrained, numbering five animals per each cage. The acoustic signal consisted of two pure tones at 1 and 6 kHz, respectively, and with equal intensity to make the total level of 125 dB SPL.
The methods for determining cochlear morphology are similar to those reported by others.72, 73 After the final ABR test, the mice were deeply anaesthetized with an overdose of ketamine, and the cochleae rapidly harvested. Surrounding soft tissues were removed and the round window and oval window were both opened. A small hole was made with a needle at the apex of the cochlea for perfusion and staining. The staining solution for succinate dehydrogenase histochemistry was freshly prepared by mixing 0.2 mol sodium succinate (2.5 ml), phosphate-buffered saline (2.5 ml) and nitro-tetranitro blue tetrazolium (5 ml). The cochlea was gently perfused through the hole at the cochlear apex and the opened round and oval windows. Following this, the cochlea was immersed in the succinate dehydrogenase solution for 45 min at 37 °C and then fixed with 10% formalin for 4 h. After fixation, the cochlea was decalcified with 5% Ethylenediaminetetraacetic acid (EDTA) solution for 3 days. The OC was dissected and surface preparations were made on slides. The cytocochleogram was determined by the spatial-percentage count of missing HCs along the cochlear duct and was established against the norm for C57 mice using custom software (as previously reported by Wang et al.74). HC loss was then measured in the prepared sections of the OC.
Histological preparations for the examination of the SGN lesion were similar to that of the cytocochleogram preparation with slight modifications. Briefly, the cochlea was perfused with 2% glutaraldehyde in phosphate-buffered saline buffer for fixation. After the perfusion, the cochlea was immersed in the fixative for 6 h at 4 °C, followed by decalcification in 5% EDTA for 3 days. The cochlea was further fixed in 1% osmium acid for 1 h at room temperature and then dehydrated in grade ethanol. Then, the sample was infiltrated with a 1:1 volume ratio of propylene oxide+Epon at room temperature overnight and transferred into 100% Epon for 4 h. Next, the sample was immersed in 100% Epon in container to be hardened in oven at 60 °C for more than 12 h. Semi-thin cross-sections of 1.5–2 μm were made along the axes of modiolus with microtome equipment and were transferred to a glass slide, stained with 1% Toluidine blue for 1 min, and then examined under a light microscope. The number of SGN cell bodies was counted in the Rosenthal canal at four locations corresponding to turns along the cochlear duct (two positions each for the basal and apical turns of the cochlea; four positions in total, see Figure 4). At each location, 10 sections were taken to cover a distance over 0.4 mm and the SGN cell body counts averaged from the 10. The numbers of auditory nerve fibers (dendrites from the SGN) were also counted in the sections crossing the habenular perforatae. For this measure, dendrites in the habenular perforatae immediately proximal to the Rosenthal canal were quantified. The number of nerve fibers was averaged from 10 habenular perforatae in each turn for each ear. The counting of nerve fibers (dendrites originating from SGNs) and SGN cell bodies was carried out using the cell counter function of ImageJ software (NIH, Rockville Pike Bethesda, MD, USA).
Western blotting analysis was used to quantify the levels of both endogenous XIAP (WT) and human XIAP derived from the ub-XIAP transgene (TG mice) in the cochlea. Electrophoresis was performed in the western blot apparatus to separate the proteins in a membrane according to their mass. For this electrophoresis analysis, proteins were extracted from the soft tissue of the cochlea and a piece of brain, measuring 2 mm3, from every animal using a standard protocol. Tissues were homogenized in RIPA buffer (1% Triton X-100, 1% SDS, 8.77% NaCl, 2.42 Tris-HCL base and 5% Deoxycholic acid, pH8) and then centrifuged at 14 000 g for 10 min at 4 °C. Supernatants were transferred to a new 1.5-ml tube. Protein concentrations were estimated using Bio-Rad (Hercules, CA, USA) reagent and a microplate reader (Elx 800 UV, Bio-tek Instrument Inc., Winooski, VT, USA). Next, 20 μg of protein from each sample was transferred into a tube containing RIPA, 2 × SDS sample buffer (7.5 μl each) and DTT (15 mg ml−1). The sample was then separated by 10–15% SDS-polyacrylamide gel electrophoresis in running buffer, then transferred to PDVF membrane. The membrane was blocked in a blocking solution (containing 1 mol Tris-HCL 25 ml, 1 mol NaCl 150 ml and Tween-20 500 μl, 5% non-fat milk powder in 1 L) overnight at 4 °C. After the proteins were separated, they are transferred to Whatman paper and incubated in a solution containing the primary antibody that recognizes a region conserved in both mouse XIAP (WT) and human XIAP (1:1500, XIAP antibody mouse, from BD Biosciences, Sparks, MD, USA). After adequate rinsing, the membrane was incubated with the secondary antibody (anti-mouse IgG horseradish peroxidase-linked antibody, 1:10 000, from Vector Laboratories, PI-2000, Burlingame, CA, USA) for a minimum of 1 h. This secondary antibody will cause the targeted bands to be colored in the membrane. The XIAPs (human and mouse) were then quantified by analyzing the bands on the membrane using a method of density analysis in six WT and six TG mice.
The primary focus of the data analysis was to determine whether XIAP overexpression reduced NIHL and cochlear cell death (namely, HCs and SGNs) normally associated with acoustic trauma. For this purpose, we first compared the shift of hearing thresholds measured by ABR between WT and TG mice by an analysis of covariance. Genotype was a fixed factor and frequency a continuous covariate. When a result was statistically significant at the level of α<0.05 or greater, post hoc tests were performed using t-test with the Bonferroni correction. ABR testing was used to verify whether XIAP overexpression reduced hearing loss produced by intense acoustic overstimulation, 1 and 4 weeks after the acoustic overstimulation. HC loss was evaluated by performing cytocochleograms. We did not focus on the pattern of cell death but performed a spatial percentage HC count. Unpaired t-tests were used to evaluate potential differences between the total HC loss for WT and TG groups. SGN and nerve fiber counts were compared across three groups of samples (no-noise control and noise-damaged cochleae from both TG and WT mice) using a one-way analysis of variance. Post hoc tests (t-test) were considered significant if α<0.05. Group data were presented in the form of the mean±s.e.m.
Auditory Brainstem Response
Inhibitor of Apoptosis Protein
Inner Hair Cell
Outer Hair Cell
Noise-Induced Hearing Loss
Organ of Corti
Spiral Ganglion Neuron
Ubiquitous X-linked Inhibitor of Apoptosis Protein
X-linked Inhibitor of Apoptosis Protein
Daniel E . Noise and hearing loss: a review. J Sch Health 2007; 77: 225–231.
Bohne B . Mechanisms of noise damage in the inner ear, In: Henderson D, Hamernik RP, Dosanjh DS, Mills JH (eds). Effects of Noise on Hearing. Raven Press: New York, 1976, pp. 41–68.
Liberman MC . Quantitative assessment of inner ear pathology following ototoxic drugs or acoustic trauma. Toxicol Pathol 1990; 18: 138–148.
Wang Y, Hirose K, Liberman MC . Dynamics of noise-induced cellular injury and repair in the mouse cochlea. J Assoc Res Otolaryngol 2002; 3: 248–268.
Chen GD, Zhao HB . Effects of intense noise exposure on the outer hair cell plasma membrane fluidity. Hear Res 2007; 226: 14–21.
Kujawa SG, Liberman MC . Adding insult to injury: cochlear nerve degeneration after ‘temporary’ noise-induced hearing loss. J Neurosci 2009; 29: 14077–14085.
Suzuki M, Yamasoba T, Ishibashi T, Miller JM, Kaga K . Effect of noise exposure on blood-labyrinth barrier in guinea pigs. Hear Res 2002; 164: 12–18.
Masutani H, Nakai Y, Kato A . Microvascular disorder of the stria vascularis in endolymphatic hydrops. Acta Otolaryngol Suppl 1995; 519: 74–77.
Yamane H, Nakai Y, Konishi K, Sakamoto H, Matsuda Y, Iguchi H . Strial circulation impairment due to acoustic trauma. Acta Otolaryngol 1991; 111: 85–93.
Smith DI, Lawrence M, Hawkins Jr JE . Effects of noise and quinine on the vessels of the stria vascularis: an image analysis study. Am J Otolaryngol 1985; 6: 280–289.
Hukee MJ, Duvall III AJ . Cochlear vessel permeability to horseradish peroxidase in the normal and acoustically traumatized chinchilla: a reevaluation. Ann Otol Rhinol Laryngol 1985; 94: 297–303.
Nicotera TM, Hu BH, Henderson D . The caspase pathway in noise-induced apoptosis of the chinchilla cochlea. J Assoc Res Otolaryngol 2003; 4: 466–477.
Bohne BA, Harding GW, Lee SC . Death pathways in noise-damaged outer hair cells. Hear Res 2007; 223: 61–70.
Yang WP, Henderson D, Hu BH, Nicotera TM . Quantitative analysis of apoptotic and necrotic outer hair cells after exposure to different levels of continuous noise. Hear Res 2004; 196: 69–76.
Hu BH, Guo W, Wang PY, Henderson D, Jiang SC . Intense noise-induced apoptosis in hair cells of guinea pig cochleae. Acta Otolaryngol 2000; 120: 19–24.
Hu BH, Henderson D, Nicotera TM . Involvement of apoptosis in progression of cochlear lesion following exposure to intense noise. Hear Res 2002; 166: 62–71.
Hu BH, Henderson D, Nicotera TM . Extremely rapid induction of outer hair cell apoptosis in the chinchilla cochlea following exposure to impulse noise. Hear Res 2006; 211: 16–25.
Eldadah BA, Faden AI . Caspase pathways, neuronal apoptosis, and CNS injury. J Neurotrauma 2000; 17: 811–829.
Miller DK . The role of the Caspase family of cysteine proteases in apoptosis. Semin Immunol 1997; 9: 35–49.
Nicholson DW, Thornberry NA . Caspases: killer proteases. Trends Biochem Sci 1997; 22: 299–306.
Van De Water TR, Lallemend F, Eshraghi AA, Ahsan S, He J, Guzman J et al. Caspases, the enemy within, and their role in oxidative stress-induced apoptosis of inner ear sensory cells. Otol Neurotol 2004; 25: 627–632.
Zimmermann KC, Bonzon C, Green DR . The machinery of programmed cell death. Pharmacol Ther 2001; 92: 57–70.
Deveraux QL, Roy N, Stennicke HR, Van Arsdale T, Zhou Q, Srinivasula SM et al. IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. Embo J 1998; 17: 2215–2223.
Deveraux QL, Takahashi R, Salvesen GS, Reed JC . X-linked IAP is a direct inhibitor of cell-death proteases. Nature 1997; 388: 300–304.
Roy N, Deveraux QL, Takahashi R, Salvesen GS, Reed JC . The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases. Embo J 1997; 16: 6914–6925.
Kominsky DJ, Bickel RJ, Tyler KL . Reovirus-induced apoptosis requires mitochondrial release of Smac/DIABLO and involves reduction of cellular inhibitor of apoptosis protein levels. J Virol 2002; 76: 11414–11424.
Ishigaki S, Liang Y, Yamamoto M, Niwa J, Ando Y, Yoshihara T et al. X-Linked inhibitor of apoptosis protein is involved in mutant SOD1-mediated neuronal degeneration. J Neurochem 2002; 82: 576–584.
Schoemaker MH, Ros JE, Homan M, Trautwein C, Liston P, Poelstra K et al. Cytokine regulation of pro- and anti-apoptotic genes in rat hepatocytes: NF-kappaB-regulated inhibitor of apoptosis protein 2 (cIAP2) prevents apoptosis. J Hepatol 2002; 36: 742–750.
Suzuki Y, Nakabayashi Y, Takahashi R . Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death. Proc Natl Acad Sci USA 2001; 98: 8662–8667.
Simons M, Beinroth S, Gleichmann M, Liston P, Korneluk RG, MacKenzie AE et al. Adenovirus-mediated gene transfer of inhibitors of apoptosis protein delays apoptosis in cerebellar granule neurons. J Neurochem 1999; 72: 292–301.
Deveraux QL, Stennicke HR, Salvesen GS, Reed JC . Endogenous inhibitors of caspases. J Clin Immunol 1999; 19: 388–398.
Deveraux QL, Reed JC . IAP family proteins—suppressors of apoptosis. Genes Dev 1999; 13: 239–252.
Trapp T, Korhonen L, Besselmann M, Martinez R, Mercer EA, Lindholm D . Transgenic mice overexpressing XIAP in neurons show better outcome after transient cerebral ischemia. Mol Cell Neurosci 2003; 23: 302–313.
Xu D, Bureau Y, McIntyre DC, Nicholson DW, Liston P, Zhu Y et al. Attenuation of ischemia-induced cellular and behavioral deficits by X chromosome-linked inhibitor of apoptosis protein overexpression in the rat hippocampus. J Neurosci 1999; 19: 5026–5033.
Crocker SJ, Liston P, Anisman H, Lee CJ, Smith PD, Earl N et al. Attenuation of MPTP-induced neurotoxicity and behavioural impairment in NSE-XIAP transgenic mice. Neurobiol Dis 2003; 12: 150–161.
Spongr V, Walton JP, Frisina RD, Kazee AM, Flood DG, Salvi RJ . Hair cell loss and synaptic loss in inferior colliculus of C57BL/6 mice: Relationship to abnormal temporal processing. In: Syka J (ed). Auditory Signal Processing in the Central Auditory Pathway. Plenum Publishing Corp.: New York, 1997.
Briner W, Willott JF . Ultrastructural features of neurons in the C57BL/6J mouse anteroventral cochlear nucleus: young mice versus old mice with chronic presbycusis. [Review] [29 refs]. Neurobiology of Aging 1989; 10: 295–303.
Spongr VP, Flood DG, Frisina RD, Salvi RJ . Quantitative measure of hair cell loss in CBA and C57BL/6 mice throughout their life spans. J Acoust Soc Am 1997; 101: 3546–3553.
Willott JF, Parham K, Hunter KP . Response properties of inferior colliculus neurons in middle-aged C57BL/6J mice with presbycusis. Hear Res 1988; 37: 15–27.
Wang J, Menchenton T, Yin S, Yu Z, Bance M, Morris DP et al. Over-expression of X-linked inhibitor of apoptosis protein slows presbycusis in C57BL/6J mice. Neurobiol Aging 2010; 31: 1238–1249.
Henderson D, Bielefeld EC, Harris KC, Hu BH . The role of oxidative stress in noise-induced hearing loss. Ear Hear 2006; 27: 1–19.
Pantano C, Reynaert NL, van der Vliet A, Janssen-Heininger YM . Redox-sensitive kinases of the nuclear factor-kappaB signaling pathway. Antioxid Redox Signal 2006; 8: 1791–1806.
Le Prell CG, Yamashita D, Minami SB, Yamasoba T, Miller JM . Mechanisms of noise-induced hearing loss indicate multiple methods of prevention. Hear Res 2007; 226: 22–43.
Raff MC . Social controls on cell survival and cell death. Nature 1992; 356: 397–400.
Frisch SM, Screaton RA . Anoikis mechanisms. Curr Opin Cell Biol 2001; 13: 555–562.
Ahmad M, Bohne BA, Harding GW . An in vivo tracer study of noise-induced damage to the reticular lamina. Hear Res 2003; 175: 82–100.
Hu B, Henderson D, Nicotera TM . Can detachment of OHCs following exposure to impulse noise cause rapid apoptosis. Association for Research in Otolaryngology, Mid-Winter Meeting. Daytona Beach, Florida, USA, 2004.
Resch U, Schichl YM, Sattler S, de Martin R . XIAP regulates intracellular ROS by enhancing antioxidant gene expression. Biochem Biophys Res Commun 2008; 375: 156–161.
Hou F, Wang S, Zhai S, Hu Y, Yang W, He L . Effects of alpha-tocopherol on noise-induced hearing loss in guinea pigs. Hear Res 2003; 179: 1–8.
Hight NG, McFadden SL, Henderson D, Burkard RF, Nicotera T . Noise-induced hearing loss in chinchillas pre-treated with glutathione monoethylester and R-PIA. Hear Res 2003; 179: 21–32.
Hu BH, Zheng XY, McFadden SL, Kopke RD, Henderson D . R-phenylisopropyladenosine attenuates noise-induced hearing loss in the chinchilla. Hear Res 1997; 113: 198–206.
Quirk WS, Shivapuja BG, Schwimmer CL, Seidman MD . Lipid peroxidation inhibitor attenuates noise-induced temporary threshold shifts. Hear Res 1994; 74: 217–220.
Seidman MD, Shivapuja BG, Quirk WS . The protective effects of allopurinol and superoxide dismutase on noise-induced cochlear damage. Otolaryngol Head Neck Surg 1993; 109: 1052–1056.
Sergi B, Fetoni AR, Paludetti G, Ferraresi A, Navarra P, Mordente A et al. Protective properties of idebenone in noise-induced hearing loss in the guinea pig. Neuroreport 2006; 17: 857–861.
Wang J, Pignol B, Chabrier PE, Saido T, Lloyd R, Tang Y et al. A novel dual inhibitor of calpains and lipid peroxidation (BN82270) rescues the cochlea from sound trauma. Neuropharmacology 2007; 52: 1426–1437.
Wang J, Ruel J, Ladrech S, Bonny C, van de Water TR, Puel JL . Inhibition of the c-Jun N-terminal kinase-mediated mitochondrial cell death pathway restores auditory function in sound-exposed animals. Mol Pharmacol 2007; 71: 654–666.
Wang J, Van De Water TR, Bonny C, de Ribaupierre F, Puel JL, Zine A . A peptide inhibitor of c-Jun N-terminal kinase protects against both aminoglycoside and acoustic trauma-induced auditory hair cell death and hearing loss. J Neurosci 2003; 23: 8596–8607.
Harris KC, Hu B, Hangauer D, Henderson D . Prevention of noise-induced hearing loss with Src-PTK inhibitors. Hear Res 2005; 208: 14–25.
Bielefeld EC, Hynes S, Pryznosch D, Liu J, Coleman JK, Henderson D . A comparison of the protective effects of systemic administration of a pro-glutathione drug and a Src-PTK inhibitor against noise-induced hearing loss. Noise Health 2005; 7: 24–30.
Borg E . Loss of hair cells and threshold sensitivity during prolonged noise exposure in normotensive albino rats. Hear Res 1987; 30: 119–126.
Chen GD, Fechter LD . The relationship between noise-induced hearing loss and hair cell loss in rats. Hear Res 2003; 177: 81–90.
Chen GD, Liu Y . Mechanisms of noise-induced hearing loss potentiation by hypoxia. Hear Res 2005; 200: 1–9.
Kujawa SG, Liberman MC . Acceleration of age-related hearing loss by early noise exposure: evidence of a misspent youth. J Neurosci 2006; 26: 2115–2123.
Canlon B . The effect of acoustic trauma on the tectorial membrane, stereocilia, and hearing sensitivity: possible mechanisms underlying damage, recovery, and protection. [Review] [168 refs]. Scandinavian Audiology. Supplementum 1988; 27: 1–45.
Chen GD . Prestin gene expression in the rat cochlea following intense noise exposure. Hear Res 2006; 222: 54–61.
Zhao HB, Santos-Sacchi J . Auditory collusion and a coupled couple of outer hair cells. Nature 1999; 399: 359–362.
Liu XZ, Ouyang XM, Xia XJ, Zheng J, Pandya A, Li F et al. Prestin, a cochlear motor protein, is defective in non-syndromic hearing loss. Hum Mol Genet 2003; 12: 1155–1162.
Zheng J, Long KB, Matsuda KB, Madison LD, Ryan AD, Dallos PD . Genomic characterization and expression of mouse prestin, the motor protein of outer hair cells. Mamm Genome 2003; 14: 87–96.
Zheng J, Richter CP, Cheatham MA . Prestin expression in the cochlea of the reeler mouse. Neurosci Lett 2003; 347: 13–16.
Zheng J, Shen W, He DZ, Long KB, Madison LD, Dallos P . Prestin is the motor protein of cochlear outer hair cells. Nature 2000; 405: 149–155.
Wang J, Menchenton T, Yin SK, Yu Z, Bance M, Mories DP et al. Over-expression of X-linked inhibitor of apoptosis protein slows presbycusis in C57BL/6J mice. Neurobiol Aging 2010; 31: 1238–1249.
Ding DL, McFadden SL, Wang J, Hu BH, Salvi RJ . Age- and strain-related differences in dehydrogenase activity and glycogen levels in CBA and C57 mouse cochleas. Audiol Neurootol 1999; 4: 55–63.
Ding D, McFadden S, Salvi R . Cochlear hair cell densities and inner-ear staining techniques. In: Willott JF (ed). Handbood of Mouse Auditory Research-From Behavior to Molecular Biology. CRC Press: New York, 2001, pp 189–204.
Wang J, Ding D, Salvi RJ . Carboplatin-induced early cochlear lesion in chinchillas. Hear Res 2003; 181: 65–72.
This thesis project was supported by the research grant of Canadian Institute of Health Research (MOP79452), the Program of Shanghai Subject Chief Scientist (08XD1403100) and the Special Program for Key Basic Research of the Ministry of Science and Technology of China (2009CB526504). We thank Dr Kiefte for his assistance in data analysis.
The authors declare no conflict of interest.
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Wang, J., Tymczyszyn, N., Yu, Z. et al. Overexpression of X-linked inhibitor of apoptosis protein protects against noise-induced hearing loss in mice. Gene Ther 18, 560–568 (2011). https://doi.org/10.1038/gt.2010.172
- X-linked inhibitor of apoptosis protein
- noise-induced hearing loss
- acoustic overstimulation
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