Mechanisms of hearing loss after blast injury to the ear

Abstract

Given the frequent use of improvised explosive devices (IEDs) around the world, the study of traumatic blast injuries is of increasing interest. The ear is the most common organ affected by blast injury because it is the body's most sensitive pressure transducer. We fabricated a blast chamber to re-create blast profiles similar to that of IEDs and used it to develop a reproducible mouse model to study blast-induced hearing loss. The tympanic membrane was perforated in all mice after blast exposure and found to heal spontaneously. Micro-computed tomography demonstrated no evidence for middle ear or otic capsule injuries; however, the healed tympanic membrane was thickened. Auditory brainstem response and distortion product otoacoustic emission threshold shifts were found to be correlated with blast intensity. As well, these threshold shifts were larger than those found in control mice that underwent surgical perforation of their tympanic membranes, indicating cochlear trauma. Histological studies one week and three months after the blast demonstrated no disruption or damage to the intra-cochlear membranes. However, there was loss of outer hair cells (OHCs) within the basal turn of the cochlea and decreased spiral ganglion neurons (SGNs) and afferent nerve synapses. Using our mouse model that recapitulates human IED exposure, our results identify that the mechanisms underlying blast-induced hearing loss does not include gross membranous rupture as is commonly believed. Instead, there is both OHC and SGN loss that produce auditory dysfunction.

Figure 1. Blast wave characteristics, measured without a mouse in the tube.

(A) The reflected and static pressures were measured to describe the blast wave profile. The stagnation pressure was measured with the pressure sensor as shown in Figure 1D (facing the oncoming blast wave). The static pressure was measured with the pressure sensor turned 90°, so that the sensing surface was facing vertically (side-on to the blast wave). Note the rapid onset of the blast wave at 0 ms, the blast wind peak about 2–3 ms later, and then the under-pressure from ∼6–20 ms. These waveforms are characteristic of that seen with an explosive detonation. The arrows highlight reflected waves that occurred outside of the blast chamber. The inset is an average power spectral density analysis of the stagnation pressure from five blast waves. (B) Varying the pressure in the reservoir chamber changed the blast wave profile. (C) There was a linear relationship between the reservoir chamber (tank) pressure and the peak blast pressure. (D) Larger magnitude blasts produced slightly longer blast durations, consistent with a longer blast wind.

Figure 2. ABR and DPOAE thresholds across the frequency spectrum for cohorts of mice exposed to different blast peak pressures.

The blast peak pressure is given in the lower right of each plot. (A,B) The lowest blast pressure cohort had a nearly complete recovery of ABR thresholds and a partial recovery of DPOAE thresholds within two weeks. However there were still statistically significant differences between the ABR and DPOAE thresholds before the blast compared to 14 days after blast (two-way ANOVA, p<0.001 for both sets of curves). (C,D) By 14 days, the middle blast pressure cohort had less recovery of ABR thresholds and almost no recovery of DPOAE thresholds (two-way ANOVA, p<0.001). (E,F) The highest blast pressure cohort had ABR and DPOAE thresholds that were even larger, and by 14 days remained higher than the thresholds before the blast (two-way ANOVA, p<0.001). (G,H) The average of the ABR and DPOAE thresholds at 16, 23, and 32 kHz (the most sensitive frequencies) two weeks after the blast. Higher peak blast pressures led to higher thresholds for both ABR and DPOAE averages (one-way ANOVA, p<0.001).

Figure 3. Perforation of the tympanic membrane after blast exposure.

Each image is from a different mouse. (A) Immediately following a blast, perforations were always seen as in this representative example (green arrows). The malleus (m) is identified. To estimate the size of the perforation, the tympanic membrane was considered to have four quadrants (inset) each containing 25%. In this example, the perforation was estimated to be 40% of the surface area of the tympanic membrane. (B) By 14 days after the blast, it was typical for the perforations to be partially healed. The original edges of the perforation (green arrows) and a portion of the perforation that had not healed (blue asterisk) can be seen. (C) By 28 days after the blast, most of the perforation had healed. The original edges of the perforation in this representative example are highlighted (green arrows). (D) There was no difference in the size of the perforation between different peak blast pressures (n = 10 for each group; one-way ANOVA, p>0.1). (E) Micro-CT of the mouse demonstrates that most of the head was scanned. (F) To assess for skull fractures, the image contrast and brightness were adjusted to remove the soft tissues. (G) The temporal bone was enlarged, demonstrating the turns of the cochlea (purple lines). The cochlear apex and base are identified. Malleus (m). No fractures of the skull or otic capsule bone were ever noted (n = 10). (H) A coronal cross-section through a representative control (i.e. age-matched) mouse temporal bone. The blue line traverses the tympanic membrane. Malleus (m); cochlea (c). (I) A representative image from a mouse three months after blast exposure demonstrates a thickening of the inferior portion of the tympanic membrane. (J) The signal density profile of the tympanic membranes shown in (H) and (I). The thickness was calculated as the width at half-maximum (green lines). (K) The tympanic membrane (TM) thickness and peak density were higher in blast-exposed mice compared to controls (Student's non-paired t-test, p<0.001 for each measure). (L,M) Long-term changes in ABR and DPOAE thresholds. One cohort of ten mice was exposed to the highest blast pressure (blast) and another cohort of mice underwent surgical perforation of their tympanic membranes (perforation). Auditory thresholds were repeatedly measured for 70 days in both cohorts. The average of the ABR and DPOAE thresholds at 16, 23, and 32 kHz in both cohorts stabilized after 28 days. However, thresholds remained higher in the blast-exposed mice (non-paired T-test, p<0.001 for each measure), consistent with permanent cochlear damage.

Figure 4. Representative plastic-embedded cochlear sections of age-matched controls and seven days after blast and.

The sections were 10 µm thick. (A) The complete cochlear cross-sections are shown with labels indicating the areas that are enlarged. (B,C,D) Enlargements of the apical turn (B), the upper basal turn (C), and the lower basal turn (D). There was no evidence for obvious gross disruption of the intracochlear soft tissues. However, there was apparent loss of OHCs in the basal region of the cochlea as assessed by loss of their dark-stained nuclei (compare arrows in D). Scale bars: A-250 µm, B-50 µm.

Figure 5. Representative whole mount preparations of the cochlear epithelium immunolabeled for prestin and myosin VIIa.

OHCs are red and IHCs are green. (A) An age-matched control mouse demonstrates the full complement of OHCs and IHCs. Scale bar 100 µm. (B) Three months after blast-exposure, substantial OHC loss was found within the basal turn. While some IHCs were missing, most were present. The transition zone roughly 30% up from the base of the cochlea (arrow) marked the point at which some OHCs were able to survive the blast trauma. (C) Cytocochleograms were performed for quantification in mice three months after blast. (D) There were no differences in the patterns of OHC loss between the three rows in mice after blast.

Figure 6. Confocal imaging of phalloidin-stained cochleae.

Residual OHCs seven days after blast exposure do not have gross disturbances of their stereociliary bundles. Asterisks indicate missing OHCs. (A,B) The apex of the cochlea. (C,D) The middle of the cochlea. (E, F) The base of the cochlea. While blast-exposed mice did not have any residual OHCs present within the far base of their cochlea, shown here is a cluster of residual OHCs at the transition zone (arrow in Fig. 5B). (G, H) Enlargements of the OHCs indicated by the white boxes in parts C&D. In all images, the normal stereociliary bundle morphology is seen. The scale bar is 8 µm.

Figure 7. Spiral ganglion neurons.

(A) Plastic embedded sections of control mice and mice seven days after blast exposure. There was a significant reduction of spiral ganglion neurons (SGNs) in blast-exposed mice compared to controls (non-paired Student's t-test, p = 0.013). SGNs were identified by their larger nuclei and prominent nucleoli. (B) Summed confocal Z-stack images of the apical turn of the cochlea in control and mice seven days after blast exposure. The number of synaptic ribbons (red punctate labeling) under the IHCs and the OHCs was reduced after blast exposure (non-paired Student's t-test, p<0.0001 for both). (C) Representative paraffin embedded cochlear cross-sections stained with DAPI from a control mouse, a mouse one day after blast exposure, and a mouse seven days after blast exposure. The boxes highlight the modiolus, which was expanded for the immunolabeling studies in (D). (D) IBA1 expression was stronger in mice one and seven days after blast exposure. Scale bars: A-50 µm, B-20 µm, C-200 µm, D-200 µm.