Age-related loss of outer hair cells was increased by OC lesion, but not by chronic conductive hearing loss.

<p>Mean survival (± SEMs) of outer hair cells (A) and inner hair cells (B) as a function of cochlear location. Cochleas were harvested at 64 wks. Groups and group sizes are as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142341#pone.0142341.g002" target="_blank">Fig 2</a>. Key in A also applies to B.</p

Threshold shifts (A,B) and changes in suprathreshold amplitude (C,D) for ABR (A,C) and DPOAEs (B,D), as measured at 64 wks and normalized to mean age-matched controls.

<p>Means (±SEMs) are shown. Amplitudes are computed by extracting the mean response for each ear at each stimulus frequency for stimulus levels from 60–80 dB SPL, inclusive. Groups and group sizes are as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142341#pone.0142341.g002" target="_blank">Fig 2</a>. Key in C applies to all panels. Downward arrows indicate that the DPOAE thresholds were at or near the measurement ceiling, thus response changes are a minimum estimate.</p

Schematics of the auditory periphery, including the TM, the cochlea and the brainstem locations of the cell bodies of the medial (M) and lateral (L) olivocochlear (OC) neurons.

<p>A: Conductive hearing loss was produced by unilateral resection of the TM (<i>TMx</i>). B: Cochlear de-efferentation was produced by a stereotaxic section of the olivocochlear bundle in the dorsal surface of the brainstem (<i>OCx</i>).</p

Age-related threshold shifts are exacerbated by ipsilateral OC lesion (<i>OCx</i>), TM removal (<i>TMx</i>) or otitis media (<i>OM</i>).

<p>Threshold shift in each group, at each age, was defined <i>re</i> mean values at the same test frequency in 8-wk controls. Each point shows mean threshold shift (±SEM) for either ABRs (A) or DPOAEs (B) for frequencies from 5–45 kHz inclusive. Group sizes are: Controls n = 11, <i>OCx</i> n = 6; <i>TMx</i> n = 10; <i>OM</i> n = 2. <i>OCx</i> cases were exclusively those where the degree of de-efferentation was greater than 75% at all cochlear regions, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142341#pone.0142341.g006" target="_blank">Fig 6</a>. Key in A applies to both panels. Downward arrows indicate that some DPOAE thresholds were at the measurement ceiling, thus the shifts represent a minimum estimate.</p

Loss of efferent innervation, whether by OC lesion or chronic conductive hearing loss, compresses the spatial distribution of afferent synapses (A,B), and inner hair cells (C), along the modiolar-pillar axis; the staggered packing of adjacent hair cells, which arises from hair cell hypertrophy, is normally maximal (C) at cochlear locations where the LOC innervation is densest (D).

<p>A,B: Spatial distribution of inner hair cell afferent synapses along the modiolar-pillar axis for each experimental groups compared to age-matched controls. Synaptic location is defined as described in Methods. Mean values are shown for each group within each 1-μm location bin. Data are combined across all cochlear locations. C: Staggering of the inner hair cell row was quantified by measuring the nuclear spread (see schematic in E), as seen in yz projections of confocal z-stacks (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142341#pone.0142341.g005" target="_blank">Fig 5</a>). Mean values (±SEMs) are shown. D: Mean density of LOC innervation (±SEMs) in age-matched control ears as a function of cochlear location. Symbol keys in A,B also apply to C,D. E: Schematic illustrating the staggered positioning of inner hair cell nuclei along the modiolar pillar axis, and the measure of “nuclear spread” used to quantify it.</p

Effects of chronic conductive hearing loss (E,F,G,H) or OC lesion (I,J,K,L) on efferent and afferent innervation in the inner and outer hair cells areas, as compared to control (A,B,C,D).

<p>Each row of images (e.g. A,B) contains a pair of maximal projections of a confocal z-stack through 10–12 adjacent hair cells, viewed either in the xy (acquisition) plane (left) or the zy (digitally rotated) plane (right). Each afferent synapse in the inner hair cell area (C,G,K) appears as a closely apposed pair of red (anti-CtBP2) and green (anti-GluA2) puncta. GluA2 puncta are not visible in the outer hair cell area (A,E,I). OC terminals in both inner and outer hair cell areas appear in the blue (anti-VAT) channel. Positions of inner hair cell nuclei are shown as dotted white circles—as seen in the red channel by adjusting gamma (not shown). The white lines in D show the modiolar-pillar and habenular-cuticular axes used in the spatial analysis of inner hair cell synapses (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142341#pone.0142341.g009" target="_blank">Fig 9</a>). Orientation of inner hair cells in the zy plane (D,H,L) is as schematized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142341#pone.0142341.g009" target="_blank">Fig 9</a>, as are the outer hair cells (B,F,J). Scale in C applies to all micrographs, which are from the 22 kHz region.</p

The relation between the degree of de-efferentation in the inner hair cell area and loss of afferent synapses (A) or ABR Wave 1 amplitude (B) is similar whether the de-efferentation is caused by OC lesion or by chronic conductive hearing loss.

<p>Data include all frequency regions from 8 to 45 kHz. Data are normalized to the mean value for age-matched controls at the appropriate frequency region. Wave 1 amplitudes (B) are extracted for stimulus levels from 60–80 dB SPL, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142341#pone.0142341.g003" target="_blank">Fig 3</a>. Data are not shown for <i>TMx ipsi</i> and <i>OM ipsi</i> groups since conductive hearing loss, <i>per se</i> profoundly decreases ABR amplitudes. Groups and group sizes are as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142341#pone.0142341.g002" target="_blank">Fig 2</a>. Key in A also applies to B.</p

Chronic conductive hearing loss, by either TM removal or otitis media, reduces OC innervation, though not as completely as can be achieved by OC lesion.

<p>A,B: Comparison of LOC and MOC density in cochlear regions ipsilateral (red, magenta) or contralateral (blue) to olivocochlear lesion (<i>OCx</i>) or otitis media (<i>OM</i>) (A) or tympanic membrane removal (<i>TMx</i>) (B). Each point is a different cochlear region from a different case (8–45 kHz regions are shown). Data from each cochlear region are normalized to mean values from age-matched controls (gray). Dashed box in A delineates 75% de-efferentation for both MOC and LOC: only cases for which all cochlear regions are within that box are included in the <i>OCx</i> group. C,D: Mean LOC and MOC innervation densities (± SEMs), respectively, as a function of cochlear frequency for the different experimental groups, normalized to control means. Keys in A and B also apply to C and D.</p

Word recognition performance was significantly poorer in the high-risk group in the presence of noise, or time compression plus reverberation.

<p>A: Mean word-recognition scores for 50 NU-6 words presented monaurally at 35 dB HL either (left) in <i>Quiet</i>, or (middle) with ipsilateral white noise at signal-to-noise ratios of 5 dB or 0 dB (<i>Noise</i>), or (right) with time compression of 45% or 65% and a reverberation time of 0.3 sec (<i>Fast + Rev</i>.). B: Same data as A, but separated by sex as indicated. All data are means (±SEM). * p<0.05, **p<0.01, ***p<0.001.</p

Speech scores were better correlated with the SP/AP ratio (A) than with the mean high-frequency threshold elevation (B).

<p>Data are the same as those from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162726#pone.0162726.g003" target="_blank">Fig 3</a>, except that values for each subject are shown. Dashed lines show regression lines for those correlations that were statistically significant. * p<0.05, **p<0.01, ***p<0.001.</p