Noise-induced cochlear synaptopathy in rhesus monkeys (Macaca mulatta)

Abstract

Cochlear synaptopathy can result from various insults, including acoustic trauma, aging, ototoxicity, or chronic conductive hearing loss. For example, moderate noise exposure in mice can destroy up to ∼50% of synapses between auditory nerve fibers (ANFs) and inner hair cells (IHCs) without affecting outer hair cells (OHCs) or thresholds, because the synaptopathy occurs first in high-threshold ANFs. However, the fiber loss likely impairs temporal processing and hearing-in-noise, a classic complaint of those with sensorineural hearing loss. Non-human primates appear to be less vulnerable to noise-induced hair-cell loss than rodents, but their susceptibility to synaptopathy has not been studied. Because establishing a non-human primate model may be important in the development of diagnostics and therapeutics, we examined cochlear innervation and the damaging effects of acoustic overexposure in young adult rhesus macaques. Anesthetized animals were exposed bilaterally to narrow-band noise centered at 2 kHz at various sound-pressure levels for 4 h. Cochlear function was assayed for up to 8 weeks following exposure via auditory brainstem responses (ABRs) and otoacoustic emissions (OAEs). A moderate loss of synaptic connections (mean of 12-27% in the basal half of the cochlea) followed temporary threshold shifts (TTS), despite minimal hair-cell loss. A dramatic loss of synapses (mean of 50-75% in the basal half of the cochlea) was seen on IHCs surviving noise exposures that produced permanent threshold shifts (PTS) and widespread hair-cell loss. Higher noise levels were required to produce PTS in macaques compared to rodents, suggesting that primates are less vulnerable to hair-cell loss. However, the phenomenon of noise-induced cochlear synaptopathy in primates is similar to that seen in rodents.

Keywords: Cochlear histopathology; Cochlear synapatopathy; Noise-induced hearing loss; Non-human primate; Permanent threshold shift; Temporary threshold shift.

Figure 1. ABR thresholds (A), threshold shifts (B) and DPOAE magnitudes (C-D) were measured before and after acoustic overexposure

A: Mean pre-exposure ABR thresholds for all animals (N=6) and for monkey M4, which received only a single 146 dB exposure. B: ABR threshold shifts measured 4-8 wks post-exposure at 108 dB SPL (teal, N=4 ears, 2 monkeys), 140 dB SPL (orange, N=2 ears, 2 monkeys), and/or 146 dB SPL (red, N=4 ears, 4 monkeys). Three of the monkeys exposed at 146 dB SPL were previously exposed to one or more lower noise levels (see Table 1). Threshold shifts for monkey M4 (exposed only at 146 dB SPL) are overlaid. Each ‘v’ represents an ear that was unresponsive to 90-dB SPL stimuli for a given frequency. C: Mean pre-exposure DPOAE magnitudes (black, N=6 monkeys) and those measured immediately post-exposure. Markers represent every other frequency step. Dashed lines represent mean noise floors. D: Mean pre-exposure DPOAE magnitudes are re-plotted for reference to those measured 4-8 wks post-exposure at 108 dB SPL (teal, N=4 ears, 2 monkeys), 140 dB SPL (orange, N=4 ears, 2 monkeys), or 146 dB SPL (red, N=8 ears, 4 monkeys). Error bars represent ±1 SEM. Asterisks represent statistical significance *P<0.05; **P<0.01; ***P<0.001. Gray bars represent the exposure band.

Figure 2. Hair-cell survival was assessed in control (A) and noise-exposed (B-C) cochleas

Micrographs of hair cells are from the 4-kHz region of a Control (A), TTS (B), and PTS (C) ear. Below each micrograph are mean cytocochleograms for corresponding groups. Supernumerary OHCs (white arrows in A, B) were excluded from analyses. Cytocochleograms for monkey M4 (146 dB SPL only) are shown for the right ear, with OHC survival averaged over the 3 rows. Error bars represent ±1 SEM. Legend applies to all panels. Gray bars represent the exposure band.

Figure 3. Hair cells and their afferent synapses were visualized by immunohistochemistry

Presynaptic ribbons (CtBP2, red), post-synaptic glutamate-receptor subunits (GluA2, green), hair cells (myo7a; gray), and nerve fibers (NFH, blue) were immunolabeled for confocal microscopy. A-A″: Maximum-intensity projections of confocal z-stacks from Control, TTS, and PTS ears at the 4-kHz region and displayed in the acquisition plane. B-B″: Orthogonal projections of the z-stacks in A-A″. C-C″: Thumbnail array of magnified x-y projections surrounding 12 selected synapses, taken from the z-stacks shown in A-B″. Synapses, i.e. juxtaposed CtBP2 and GluA2 puncta (yellow arrowheads), and “orphan“ ribbons, lacking GluA2 puncta (white arrowheads), are shown. Enlarged, hollow ribbons (C″), seen only in severely damaged regions of PTS cochleas, could be either paired or orphaned (yellow and red arrowheads in C″, respectively). Images are from an 8 yr-old male control (A-C), an 11 yr-old male with TTS (A′-C′), and a 10 yr-old male with PTS (A″-C″).

Figure 4. Synapse survival was assessed in IHCs of control (A) and noise-exposed (B) monkeys at nine cochlear regions

A: Mean synaptic counts for N=7 ears, 4 monkeys are shown by the thick line and filled symbols. Traces for individual monkeys (thin lines) show no differences between animals from the two vivaria. B: Synapse survival is computed by normalizing to the mean data in panel A. TTS (teal) and PTS (red) cochleas had significant, frequency-dependent cochlear synaptopathy. For monkey M4 (single 146-dB SPL exposure), mean synapse survival (L and R) is also plotted separately. Statistical significance is reported relative to controls: *P<0.05; **P<0.01. Error bars represent ±1 SEM. Gray bars represent the exposure band.

Figure 5. Surviving IHCs were significantly de-afferented in regions with little OHC loss

Cytocochleograms, showing IHC and OHC survival in each row (black), are plotted for left (A) and right (B) ears of a PTS monkey with asymmetric histopathology (M3). The synaptic counts per surviving IHC are overlaid (red). Gray bars represent the exposure frequency. Legend applies to both panels.

Figure 6. Pre-synaptic ribbons were enlarged in noise-exposed cochleas

Normalized volumes for all pre-synaptic ribbons (A) and the percentage of ribbons classified as “hollow” (B) in controls (black), TTS monkeys (teal), and PTS monkeys (red). Paired and orphan ribbons were included in both analyses. Gray bars represent the exposure band. Error bars represent ±1SEM. For monkey M4 (single 146-dB SPL exposure), the left and right cochleas are plotted separately. Legend applies to both panels. Asterisks represent significance (***P<0.001).

Figure 7. Cable-like myosin aggregates were common in IHCs of PTS ears

Maximum-intensity projections of confocal z-stacks in the x-z plane (A) and y-z plane (B), including 7 IHCs from the 2.8 kHz region in a PTS cochlea. Many IHCs (asterisks in A) show myosin 7a aggregates spanning the apical-basal pole. C: Mean % of affected IHCs for control (black), TTS (teal), and PTS (red) cochleas (N=7, 4, 8 cochleas, respectively). Error bars represent ±1 SEM. Gray bar represents the exposure band.

Figure 8. IHC pathologies were common in PTS ears

A-C: Confocal micrographs of pathological IHCs are marked with arrows to indicate: cytoplasmic extrusions from the cuticular plate (A), or the basal pole (B), myosin 7a-positive nuclei (C), and irregular stereocilia (C, open arrow; not quantified). The yellow dashed line in (B) traces the edges of the tunnel of Corti. Plots below each micrograph show the percentage of IHCs affected in each group: For monkey M4 (single 146-dB SPL exposure), data for each ear are plotted separately. Legend in B applies to all panels. Error bars represent ±1 SEM. Gray bars represent the exposure band.