Confocal imaging shows a reduction in CtBP2 stained presynaptic ribbons following neuropathic noise.

<p>Short-term (<b>a</b>-<b>c</b>) and long-term (<b>d</b>-<b>f</b>) loss of presynaptic ribbons in inner hair cells (IHCs) after exposure to neuropathic (97 dB SPL) noise compared to age-matched animals exposed to non-neuropathic noise (94 dB SPL) and unexposed controls. Presynaptic ribbons are labeled with anti-CtBP2 antibodies (red). Confocal images are maximal projections of z-stacks of ribbons within 4–5 IHCs in the 32 kHz region. Dashed lines depict IHC outlines. Scale bar: 10 μm (<b>a</b>-<b>f</b>).</p

Late-onset loss of cochlear neurons following neuropathic noise.

<p>Density of spiral ganglion neuron somata as a function of post exposure time, as seen in the ~11 kHz (left) and ~32 kHz (right) regions after exposure to neuropathic (97 dB SPL) or non-neuropathic (94 dB SPL) noise. Data are group means (± SD), expressed relative to data from the youngest unexposed ears. Statistical significance of the group differences is indicated by asterisks: *, <i>p < 0</i>.<i>05</i>; **, <i>p < 0</i>.<i>01</i>. N = 3–7 animals per time and noise exposure.</p

Neural response is reduced following neuropathic noise.

<p>ABR wave I peak-to-peak amplitudes (<b>a</b>) and DPOAE amplitudes (<b>b</b>) in response to 11.3 kHz tones (left column) and 32.0 kHz tones (right column) recorded from 6 hours to 16 months post exposure to noise causing PTS (100 dB SPL, purple), neuropathic noise causing TTS (97 dB SPL, blue), non-neuropathic noise causing TTS (94 dB SPL, green), and unexposed animals (red). ABR amplitudes are in response to 80 dB SPL tones (the maximum level tested) and DPOAE amplitudes are in response to 80 dB SPL tones. Noise parameters are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125160#pone.0125160.g001" target="_blank">Fig 1</a>. Statistical significance of the group differences is indicated by asterisks: *: <i>p < 0</i>.<i>05</i>, **: <i>p < 0</i>.<i>01</i>, ***: <i>p < 0</i>.<i>001</i>. Data are group means ± SD. N = 5–9 mice per time point for noise-exposed and control groups.</p

Degeneration of spiral ganglion neurons is assessed in cochlear sections after neuropathic noise.

<p>Midmodiolar sections through Rosenthal’s canal show permanent loss of spiral ganglion neurons in the apex (<b>b</b>) and base (<b>d</b>) 16 months after exposure to neuropathic noise (97 dB SPL) compared to age-matched unexposed controls (<b>a</b>, <b>c</b>). Scale bar: 50 μm (a-d).</p

Peripheral axons of cochlear nerve fibers slowly degenerate after neuropathic noise.

<p>Density of peripheral axons as a function of post exposure time in the ~11 kHz (left) and ~32 kHz (right) region, following exposure to neuropathic (97 dB SPL) or non-neuropathic (94 dB SPL) noise. Data are group means (± SD), expressed relative to data from the youngest unexposed ears. Statistical significance of the group differences is indicated by asterisks: *: <i>p < 0</i>.<i>05</i>, **: <i>p < 0</i>.<i>01</i>. N = 3–7 animals per time and noise exposure.</p

Numbers of presynaptic ribbons are reduced after neuropathic noise.

<p>Synaptic counts in inner hair cells in the 11.3 kHz (left) and 32.0 kHz (right) regions following exposure to neuropathic (97 dB SPL) or non-neuropathic (94 dB SPL) noise. Statistical significance of the group differences is indicated by asterisks: *: <i>p < 0</i>.<i>05</i>, **: <i>p < 0</i>.<i>01</i>, ***: <i>p < 0</i>.<i>001</i>. Data are group means ± SD. N = 6–7 animals per time and noise exposure.</p

Cochlear-nerve peripheral axons can be counted in tangential sections through the osseous spiral lamina.

<p>Tangential cuts though the osseous spiral lamina in the ~32 kHz region of cochleae 1 month (<b>b</b>), 8 months (<b>d</b>) or 16 months (<b>f</b>) after exposure to neuropathic noise (97 dB SPL) demonstrate loss of peripheral axons compared to age-matched unexposed controls (<b>a</b>, <b>c</b>, <b>e</b>). These sections show fascicles of cochlear-nerve peripheral axons roughly midway between their cell bodies in the spiral ganglion and their peripheral terminals in the organ of Corti. Scale bar: 10 μm (<b>a</b>-<b>f</b>).</p

Cochlear thresholds are either permanently or temporarily shifted following neuropathic noise.

<p>ABR (left column) and DPOAE (right column) thresholds resulting from exposure of 6 week old mice to 8–16 kHz noise for 2 hours at 100 dB SPL (purple), 97 dB SPL (neuropathic exposure, blue) and 94 dB SPL (non-neuropathic exposure, green) compared to unexposed controls (red). Thresholds are shown as group means ± SD. The noise band is depicted as a gray vertical bar. Time post exposure (pe) is specified in hours (h), weeks (w) or months (m) in vertical boxes on the right side of each row. N = 5–9 mice per time point and noise exposure.</p

Cochlear sensory cells are intact after neuropathic noise.

<p>Photomicrographs of the organ of Corti from the ~32 kHz region of mice exposed to 97 dB SPL noise and evaluated either 1 month (<b>a</b>) or 16 months (<b>b</b>) after exposure. Scale bar: 20 μm (<b>a</b>-<b>b</b>).</p

Auditory measurements from <i>Fgf</i> mutant mice.

<p>(A) ABR and (B) DPOAE thresholds demonstrate profound hearing loss at all frequencies in <i>Fgf</i> mice, and moderate hearing loss at high frequencies in <i>Fgf</i> mice. (C) ABR wave I amplitudes appear slightly depressed in <i>Fgf</i> mice but are not statistically differentiable (except at 32 kHz where threshold differences impact magnitude ). ABR amplitudes in <i>Fgf</i> mice are significantly reduced compared to the other genotypes . (D) Threshold adjusted ABR wave I latency measurements demonstrate significant increases in latency in both <i>Fgf</i> and <i>Fgf</i> genotypes when compared to <i>Fgf</i> littermates . Error bars present standard error of the mean.</p