Multifunctional redox modulator prevents blast-induced loss of cochlear and vestibular hair cells and auditory spiral ganglion neurons

Abstract

Blast wave exposure, a leading cause of hearing loss and balance dysfunction among military personnel, arises primarily from direct mechanical damage to the mechanosensory hair cells and supporting structures or indirectly through excessive oxidative stress. We previously reported that HK-2, an orally active, multifunctional redox modulator (MFRM), was highly effective in reducing both hearing loss and hair cells loss in rats exposed to a moderate intensity workday noise that likely damages the cochlea primarily from oxidative stress versus direct mechanical trauma. To determine if HK-2 could also protect cochlear and vestibular cells from damage caused primarily from direct blast-induced mechanical trauma versus oxidative stress, we exposed rats to six blasts of 186 dB peak SPL. The rats were divided into four groups: (B) blast alone, (BEP) blast plus earplugs, (BHK-2) blast plus HK-2 and (BEPHK-2) blast plus earplugs plus HK-2. HK-2 was orally administered at 50 mg/kg/d from 7-days before to 30-day after the blast exposure. Cochlear and vestibular tissues were harvested 60-d post-exposure and evaluated for loss of outer hair cells (OHC), inner hair cells (IHC), auditory nerve fibers (ANF), spiral ganglion neurons (SGN) and vestibular hair cells in the saccule, utricle and semicircular canals. In the untreated blast-exposed group (B), massive losses occurred to OHC, IHC, ANF, SGN and only the vestibular hair cells in the striola region of the saccule. In contrast, rats treated with HK-2 (BHK-2) sustained significantly less OHC (67%) and IHC (57%) loss compared to the B group. OHC and IHC losses were smallest in the BEPHK-2 group, but not significantly different from the BEP group indicating lack of protective synergy between EP and HK-2. There was no loss of ANF, SGN or saccular hair cells in the BHK-2, BEP and BEPHK-2 groups. Thus, HK-2 not only significantly reduced OHC and IHC damage, but completely prevented loss of ANF, SGN and saccule hair cells. The powerful protective effects of this oral MFRM make HK-2 an extremely promising candidate for human clinical trials.

Figure 1

HK-2 suppresses blast-induced hair cell loss. Representative photomicrograph of surface preparations from the middle of basal turn of the cochlea stained with Harris hematoxylin. Cochlea evaluated 2-months after exposure to 6 blasts of ~ 186 dB peak sound pressure level. Results from (A) blast-alone (B) group, (B) blast group with earplugs (BEP), (C) blast group treated with HK-2 (BHK-2) and (D) blast group treated with earplugs and HK-2 (BEPHK-2). In the B group, nearly all outer hair cells (OHC) and inner hair cells (IHC) are missing and replaced by large flat, cuboidal cells (panel A, white stars). Nearly all OHC and IHC present in blasted-exposed rats treated with ear plugs (B) or earplugs combined with HK-2 (D). Surface preparations from blast-exposed rats treated with HK-2 (C) shows patches of missing hair cells and flattened epithelium (white stars) interspersed with regions where rows of OHC and IHC are present.

Figure 2

HK-2 prevents blast-induced loss of outer hair cells (OHC) and inner hair cells (IHC). Mean (n = 6, + /− SEM) cochleograms for the B, BEP, BHK-2 and BEPHK-2 groups. Cochleograms show percent (A) OHC loss and (B) IHC loss versus percent distance from the apex of the cochlea (20% intervals). Lower bar shows rat tonotopic map. A two-way repeated measures analysis revealed a significant effect of treatment (p < 0.0001) and cochlear location (p < 0.0001); see text for details. Bonferroni post-test test revealed significant differences in the amount of OHC or IHC loss between B versus BEP group (#), B versus BHK-2 group (%), BEP versus BHK-2 group ($) and BHK-2 and BEPHK-2 group (&). 1, 2 and 3 symbols correspond to p < 0.05, 0.01 and 0.001; see text for details.

Figure 3

HK-2 suppresses blast-induced loss of auditory nerve fibers (ANF). Representative photomicrographs of toluidine blue stained, 3 µm sections from middle of basal turn of cochlea. Data obtained 2-months after exposure to 6 blasts of ~ 186 dB peak sound pressure level. Results show habenula perforata in (A) B group, dashed circle, (B) BEP group, (C) BHK-2 group, and (D) BEPHK-2 group. (E) High magnification view of habenula perforata in B group devoid of ANF versus BHK-2 group filled with toluidine blue stained ANF. ANF characterized by darkly stained ring of membrane surrounding pale cytoplasm (arrowheads). (F) Mean (n = 6, + /− SEM) number of ANF per habenula perforata in B group significantly less (p < 0.001) than in BEP group, BHK-2 group and BEPHK-2 group; see text for details.

Figure 4

HK-2 attenuates blast-induced degeneration of spiral ganglion neurons (SGN) in Rosenthal’s canal. Representative photomicrograph of toluidine blue stained sections from the middle of the basal turn of cochlea (25 kHz location) 2-months after exposure to 6 blasts of ~ 186 dB peak sound pressure level. Sections from (A) B group, (B) BEP group, (C) BHK-2 group, and (D) BEPHK-2 group. Red arrows in Fig. 4A point to a few surviving SGN in Rosenthal’s canal. (E) High magnification view of toluidine blue stained SGN soma (arrowheads). (F) Mean (n = 6, + /− SEM) number of SGN per section in B group significantly less (###, p < 0.001) than in BEP group, BHK-2 group and BEPHK-2 group; see text for details.

Figure 5

HK-2 reduces blast-induced degeneration of vestibular hair cells in saccule. (A,B) Representative photomicrographs of surface preparations of saccule in vicinity of striola; specimens stained with Harris hematoxylin. (A) Representative photomicrograph of saccule obtained from rat in the BEP group two months post-blast. White arrowheads point to darkly stained nuclei of vestibular hair cells surrounded by lightly stained nerve terminals. Number of vestibular hair cells counted in 0.12 × 0.09 mm areas (0.0108 mm²) in striola and marginal regions of saccule. (B) Representative photomicrograph from saccule of rat in blast exposed group (B) two months post-exposure. Many hair cells missing (yellow arrows) from center of striola and in adjacent circular patches in contrast to high density of vestibular hair cells in marginal area. (C) Mean (n = 6, + /− SEM) hair cell density in striola region of saccule. Hair cell density in the B group significantly less than in the BEP group (p < 0.001), BEPHK-2 group (p < 0.001) and BHK-2 group (p < 0.01). (D) Mean (n = 6, + /− SEM) hair cell density in marginal region of saccule. Hair cell densities in the four groups (B, BEP, BEPHK-2 and BHK-2) were similar and not significantly different from one another.

Figure 6

Blast wave exposure did not alter hair cell densities in the striola and marginal regions of the utricle and the ampulla of three semicircular canals. (A) Mean (n = 6, + /− SEM) vestibular hair cell densities (HC/0.0108 mm²) in the striola region of the utricle; note nearly identical values across B, BEP, BHK-2 and BEPHK-2 experimental groups. (B) (A) Mean (n = 6, + /− SEM) vestibular hair cell densities (HC/0.0108 mm²) in the marginal region of the utricle nearly identical across B, BEP, BHK-2 and BEPHK-2 experimental conditions. (C) Mean (n = 18, + /− SEM) vestibular hair cell densities (HC/0.0108 mm2) in ampulla of crista of three semicircular canals (n = 6/canal) nearly identical across B, BEP, BHK-2 and BEPHK-2 experimental conditions.

Figure 7

Earplugs (EP) provide moderately greater protection against blast-induced hair cell loss than HK-2; HK-2 combined with EP failed to provide additional protection. Four comparisons showing the percent difference in reduction of (A) OHC loss and (B) IHC loss as function of cochlear location/frequency: (a) EP vs B: EP provided the greatest reduction of blast-induced OHC and IHC loss over most of the cochlea; reduction of hair cell loss nearly constant across cochlear location/frequency except for greatly reduced IHC loss near the apex of the cochlea. (b) HK-2 vs B: HK-2 provided the second greatest reduction of OHC and IHC losses. Reduction of OHC loss greatest near low-frequency apex declining toward high-frequency base. IHC loss reductions greatest in middle/mid-frequency region of cochlea. (c) EP vs HK-2: EP provided greater reductions in OHC and IHC losses than HK-2; EP protective effect increased from low-frequency apex towards high-frequency base. (d) EP + HK-2 vs EP: No further reduction of OHC and IHC loss for EP plus HK-2 versus EP alone (i.e., lack of synergy).