Blast-induced cochlear synaptopathy in chinchillas

Abstract

When exposed to continuous high-level noise, cochlear neurons are more susceptible to damage than hair cells (HCs): exposures causing temporary threshold shifts (TTS) without permanent HC damage can destroy ribbon synapses, permanently silencing the cochlear neurons they formerly activated. While this "hidden hearing loss" has little effect on thresholds in quiet, the neural degeneration degrades hearing in noise and may be an important elicitor of tinnitus. Similar sensory pathologies are seen after blast injury, even if permanent threshold shift (PTS) is minimal. We hypothesized that, as for continuous-noise, blasts causing only TTS can also produce cochlear synaptopathy with minimal HC loss. To test this, we customized a shock tube design to generate explosive-like impulses, exposed anesthetized chinchillas to blasts with peak pressures from 160-175 dB SPL, and examined the resultant cochlear dysfunction and histopathology. We found exposures that cause large >40 dB TTS with minimal PTS or HC loss often cause synapse loss of 20-45%. While synaptopathic continuous-noise exposures can affect large areas of the cochlea, blast-induced synaptopathy was more focal, with localized damage foci in midcochlear and basal regions. These results clarify the pathology underlying blast-induced sensory dysfunction, and suggest possible links between blast injury, hidden hearing loss, and tinnitus.

Figure 1

Structure and performance of the custom shock tube. (A) Our shock tube consists of a compressed air chamber (1), separated from an expansion tube (2), and catenoidal horn (3) by a thin Mylar or brass membrane (not shown) with a remotely actuated puncture pin. In the image, the air chamber has been pulled away from the expansion tube, and no membrane is in place. The total length of the apparatus, including the horn is 6′6″. When the air chamber is filled to 27 psi, peak pressures near 175 dB SPL can be reliably produced at the orifice of the horn. (B) Example pressure waveforms measured above the animal’s head (at the midline) for levels spanning the range tested and arbitrarily aligned by placing time 0 at 5% peak pressure. (C) Example spectra obtained from pressure waveforms, as measured at the tragus, from blasts spanning the range of peak pressures. (D,E) Pressure waveforms (peak-aligned) and resultant spectra measured near the tragus during 10 consecutive blasts @ 165 dB SPL peak.

Figure 2

A large eardrum rupture seen after a single 175 dB blast. After each blast, we examined the ear canal and eardrum with an otoscope. Eardrum rupture was never seen after 160 dB or 165 dB blasts, but was sometimes seen after a single 175 dB blast (4/20 ears), and always seen after 5–10 consecutive 175 dB blasts (4/4). Ruptures were usually small tears in the inferior pars tensa, and a small amount of bleeding was often visible.

Figure 3

Low-power image of the type used to assess hair cell survival. Widefield image centered on the 13 kHz region of an ear exposed to 10 blasts @ 165 dB SPL peak. Yellow lines illustrate the boundaries used to bin the hair cell counts, corresponding to 2% increments of cochlear length. White arrows point to two regions where all three rows of OHCs are missing. OHC loss was 9.3% when averaged between the central 2% segment shown here and the segment immediately to its right: bin widths in Figs 4 and 5 are 4% of cochlear length.

Figure 4

Summary histopathology (A,C) and physiology (B) from control ears (n = 8) and cases exposed to a single blast at the lowest SPL (n = 4; 160 dB peak). Data were obtained 1 wk post exposure. Each line in each panel is from a different case. Each IHC synapse value is derived from counts in two adjacent z-stacks, divided by the number of IHCs (including fractional values) in the same stacks. Each hair cell count is derived as shown in Fig. 3. Physiological measures were not obtained from this exposed group, thus there is no Panel D. All data are normalized to control means.

Figure 5

Summary histopathology (A–C) and pathophysiology (D–F) from blast groups exposed at the three higher SPLs and obtained 1 wk post exposure. Pink shading indicates the range of control values for each metric (see Fig. 4). Line colors are used to identify individual ears within each column, allowing direct comparison of pathophysiology and histopathology in the same ear. Cases with significant synaptopathy (i.e. fewer synapses than control mean by >2 standard deviations) are shown with colored lines; grey lines are for cases without significant synaptopathy. Dashed lines are for cases with ruptured eardrum. The key in C applies to all panels. All data are normalized to control means. All other aspects of data acquisition are as described in the caption for Fig. 4.

Figure 6

Some exposed ears showed striking loss of synaptic ribbons (CtBP2-red) in localized regions. (A,C) Confocal z-stacks from the 16 kHz region of a control and an exposed ear, 1 wk post blast, shown as maximum projections in the acquisition plane (xy). (B,D) The same z-stacks reprojected into the zy plane. The exposed ear also shows stereocilia damage (espin - cyan) and disruption of the normal modiolar-pillar gradient in ribbon size, which is normally correlated with the gradient of spontaneous rate and threshold in auditory-nerve fibers.

Figure 7

Orphan ribbons, lacking apposed glutamate receptor patches, were rare in blast-exposed ears. (A) Selected synaptic complexes from the control and exposed z-stacks shown in Fig. 6. Red arrows indicate orphan ribbons. (B) The histograms compare the % orphan ribbons per z-stack for all samples from 2–22 kHz, as compiled for all control (black) and exposed (red) cases. All data were gathered at 1 wk post blast.

Figure 8

Most synaptopathic regions spanned at least 500 microns (~60 IHCs) along the cochlear spiral. For several of the synaptopathic points identified in the routine screen of half-octave positions along the cochlear spiral (e.g. Fig. 5B), we went back to the samples and imaged adjacent fields apical and basal to the initial sample. The case-identifying colors used here are the same as those in Fig. 5B; the key identifies the frequency region in question. The shaded pink region represents the combined control range from 2.3, 15, and 22 kHz.

Figure 9

The amount of temporary threshold shift (TTS) was highly variable, as shown here for two ears from two animals exposed to 10 blasts @ 165 dB SPL. (A) Threshold shifts for CAPs and DPOAEs as measured 5.5–7.5 hrs after the blast. Control range (pink shading) is from Fig. 4B. (B) Threshold shifts at 16 kHz for each of the four animals at numerous post-blast time points show the stability of threshold shifts in this acute phase. Downward arrows indicate minimum estimates because thresholds were higher than those for system-generated distortion. One ear only had CAP measures at one post-blast timepoint. All data are normalized to control means.