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. 2021 Dec;38(23):3248-3259.
doi: 10.1089/neu.2020.7556.

Correlation of Histomorphometric Changes with Diffusion Tensor Imaging for Evaluation of Blast-Induced Auditory Neurodegeneration in Chinchilla

Kathiravan Kaliyappan  1 , Johan Nakuci  2 , Marilena Preda  3   4 , Ferdinand Schweser  3   4 , Sarah Muldoon  5 , Vijaya Prakash Krishnan Muthaiah  1
Affiliations

Correlation of Histomorphometric Changes with Diffusion Tensor Imaging for Evaluation of Blast-Induced Auditory Neurodegeneration in Chinchilla

Kathiravan Kaliyappan et al. J Neurotrauma. 2021 Dec.

Abstract

In the present study, we have evaluated the blast-induced auditory neurodegeneration in chinchilla by correlating the histomorphometric changes with diffusion tensor imaging. The chinchillas were exposed to single unilateral blast-overpressure (BOP) at ∼172dB peak sound pressure level (SPL) and the pathological changes were compared at 1 week and 1 month after BOP. The functional integrity of the auditory system was assessed by auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE). The axonal integrity was assessed using diffusion tensor imaging at regions of interests (ROIs) of the central auditory neuraxis (CAN) including the cochlear nucleus (CN), inferior colliculus (IC), and auditory cortex (AC). Post-BOP, cyto-architecture metrics such as viable cells, degenerating neurons, and apoptotic cells were quantified at the CAN ROIs using light microscopic studies using cresyl fast violet, hematoxylin and eosin, and modified Crossmon's trichrome stains. We observed mean ABR threshold shifts of 30- and 10-dB SPL at 1 week and 1 month after BOP, respectively. A similar pattern was observed in DPAOE amplitudes shift. In the CAN ROIs, diffusion tensor imaging studies showed a decreased axial diffusivity in CN 1 month after BOP and a decreased mean diffusivity and radial diffusivity at 1 week after BOP. However, morphometric measures such as decreased viable cells and increased degenerating neurons and apoptotic cells were observed at CN, IC, and AC. Specifically, increased degenerating neurons and reduced viable cells were high on the ipsilateral side when compared with the contralateral side. These results indicate that a single blast significantly damages structural and functional integrity at all levels of CAN ROIs.

Keywords: auditory cortex; auditory neurodegeneration; blast-overpressure; chinchilla; cochlear nucleus; impulse noise exposure; inferior colliculus.

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Conflict of interest statement

No competing financial interests exist.

Figures

FIG. 1.
FIG. 1.
Representative impulse waveform at the tragus of the ipsilateral (A) and contralateral (B) ears. The representative frequency spectrum of the impulse waveform at the tragus of the ipsilateral (C) and contralateral (D) ears. Color image is available online.
FIG. 2.
FIG. 2.
The effect of blast-overpressure (BOP) on the amplitude of the distortion product otoacoustic emissions (DPOAE) (n = 6). The graph represents the data of DPOAE amplitudes in the ipsilateral (A) and contralateral (B) ears at 1 week post-BOP and 1 month post-BOP across various f2 frequencies. *comparison between pre-BOP and 1 month post-BOP; $comparison between pre-BOP and 1 week post-BOP; #comparison between 1 week post-BOP and 1 month post-BOP. The values with *p < 0.05; **p < 0.01; ***p < 0.001; #p < 0.05; ##p < 0.001, and $p < 0.05 are considered as significant. Color image is available online.
FIG. 3.
FIG. 3.
(A and B) shows the representative unweighted A0 image and major tensor axis direction. Images were acquired along 30 directions (b = 1000 s/mm2). On acquiring robust images, fractional anisotropy, radial diffusivity and axial diffusivity were calculated. Color image is available online.
FIG. 4.
FIG. 4.
Representative T2-weighted magnetic resonance (MR) images of axial (A–C) and coronal slices (D–F) with highlighted regions of interest (ROIs) (auditory cortex, inferior colliculus, and cochlear nucleus). Color image is available online.
FIG. 5.
FIG. 5.
The images show representative images of the cresyl fast violet (CFV) stained brain sections at the level of the (A) cochlear nucleus (CN), (B) inferior colliculus (IC), and (C) auditory cortex (AC). The middle panels indicate the ipsilateral (1 and 2) and contralateral (3 and 4) images (400 × magnification) of the CN, IC, and AC regions. The black arrows indicate the viable neurons and the red arrows indicate the darkly stained neurons in the regions of interest (ROIs). The histograms (5 and 6) indicate the viable/dark neuron counts (*p < 0.05, **p < 0.01, and ***p < 0.001 vs. control; #p < 0.05 and ###p < 0.001 vs. left side) in the respective ROIs. The data are represented as mean ± standard error of the mean (SEM). Color image is available online.
FIG. 6.
FIG. 6.
The images show representative images of the hematoxylin and eosin (H&E)-stained brain sections at the level of the (A) cochlear nucleus (CN), (B) inferior colliculus (IC), and (C) auditory cortex (AC). The right panels indicate the ipsilateral (1 and 2) and contralateral (3 and 4) images (400 × magnification) of the CN, IC, and AC regions. The black arrows indicate the viable neurons, and the red arrows indicate the darkly stained neurons in the regions of interest (ROIs). Color image is available online.
FIG. 7.
FIG. 7.
The images show representative images of the modified (Crossmon's) trichrome (MT) stained brain sections at the level of the (A) cochlear nucleus (CN), (B) Inferior colliculus (IC), and (C) auditory cortex (AC). The middle panels indicate the ipsilateral (1 and 2) and contralateral (3 and 4) images (400 × magnification) of the CN, IC, and AC regions. The white arrows indicate the viable neurons and the yellow arrows indicate the neurons undergoing apoptosis in the regions of interest (ROIs). The histograms (5) indicate the apoptotic neuron counts (***p < 0.001 vs. control; ###p < 0.001 vs. left side) in the respective ROIs. The data are represented as mean ± standard error of the mean (SEM). Color image is available online.
FIG. 8.
FIG. 8.
Blast-induced auditory brainstem response (ABR) threshold changes in chinchilla. The line graphs indicate the ABR thresholds on the (A) ipsilateral and (B) contralateral sides across various frequencies (0.5, 1, 2, 4, and 8 kHz) at pre-blast overpressure (BOP), and 1 week and 1 month post-BOP. Data are represented as mean ± standard error of the mean (SEM). Two-way analysis of variance (ANOVA) repeated measures with Bonferroni post-hoc test was performed. *comparison between pre-BOP and 1 month post -BOP; $comparison between pre-BOP and 1 week post-BOP; #comparison between 1 week post-BOP and 1 month post-BOP. The values with *p < 0.05; **p < 0.01 versus pre-BOP; $p < 0.05; $$$p < 0.001; #p < 0.05; and ###p < 0.001 are considered as significant. Color image is available online.
FIG. 9.
FIG. 9.
Effect of blast-overpressure (BOP) on the central auditory neuraxis using diffusion tensor imaging (DTI) analysis. (i) Axial diffusivity (AD) in the (A) auditory cortex, (B) inferior colliculus, and (C) cochlear nucleus at the pre-BOP, 1 week post-BOP, and 1 month post-BOP time points; (ii) radial diffusivity (RD) in the (A) auditory cortex, (B) inferior colliculus, and (C) cochlear nucleus at the pre-BOP, 1 week post-BOP, and 1 month post-BOP time points; (iii) mean diffusivity (MD) in the (A) auditory cortex, (B) Inferior colliculus, and (C) cochlear nucleus at the pre-BOP, 1 week post-BOP, and 1 month post-BOP time points; and (iv) fractional anisotropy (FA) in the (A) auditory cortex, (B) Inferior colliculus, and (C) cochlear nucleus at the pre-BOP, 1 week post-BOP, and 1 month post-BOP time points. Data are presented as the mean ± standard error of the mean (SEM).
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