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Journal of Neurotrauma logoLink to Journal of Neurotrauma
. 2021 Nov 23;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,*,**
PMCID: PMC8917894  PMID: 34605670

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

Introduction

Blast-overpressure (BOP) is responsible for 86% of United States service members wounded in action in Iraq and Afghanistan between 2001 and 2017.1,2 The positive pressure phase of brief duration BOP (> 10 kPa) inflicts mechanical damage to the cochlea and its central pathways, thereby initiating a cascade of pathological cellular processes resulting in neurodegeneration and cell death in the cochlea and higher auditory centers (e.g., cochlear nucleus [CN], inferior colliculus [IC], and auditory cortex [AC]) by activation of oxidative mechanism and neuroinflammation. The mechanisms of BOP-induced injury have been studied in different animal models such as rats, mice, pigs,3 chinchillas,4,5 and monkeys6 However, gaps remain in generalizing the pathophysiology arising from animal injury to humans.7 Similar to the BOP acoustic parameters, the choice of animal species/strain can have a substantial impact on the systemic response to the blast.8 Adhering to the guidelines for using animal models in blast research,9,10 in this study we used the chinchilla as an animal model of BOP trauma to determine pathophysiological mechanisms in the auditory pathway including the CN, IC, and AC. The results of this line of research will be useful when translating experimental findings to humans.

Here, we evaluated the structural and functional integrity of the auditory pathway using morphometric measures and diffusion tensor imaging (DTI) along with non-invasive electrophysiological hearing assays such as auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE) before and after BOP. In this study, the tissue microstructure of the auditory pathway was probed using a quantitative and non-invasive magnetic resonance DTI (MR-DTI) approach to collect diffusion-weighted images based on water diffusion in selected portions of the auditory nervous system. This helps to calculate water diffusion-tensor-related parameters such as fractional anisotropy (FA), radial diffusivity (RD), axial diffusivity (AD), and mean diffusivity (MD), which reflects the magnitude and direction of water molecule diffusion in tissues, and which are highly sensitive for detecting microscopic differences in tissue properties. Therefore, the quantitative scalar DTI metrics are compared with histomorphometric analysis using stains such as cresyl fast violet (CFV), hematoxylin and eosin (H&E), and modified (Crossmon's) trichrome (MT), and linked the cytoarchitecture and microstructural features of white matter fiber tracts in the central auditory neuraxis (CAN). The mechanism of BOP injury investigated in this study is generalizable to humans for two reasons: (1) we used chinchillas, whose frequency range of hearing is similar to that of humans and (2) the measures that are used to probe the pathophysiological changes (e.g., ABR/DPOAE, and neuroimaging) are commonly applied to humans as well.

Methods

Animals and unilateral BOP

Male chinchillas (n = 15) weighing 350–500g (Ryerson Ranch) were used for these experiments. For non-invasive, functional assays, for the intra-animal comparison of pre-BOP versus 1 week post-BOP, 12 animals were included. For non-invasive DTI, for the intra-animal comparison between time points (TPs) (pre-BOP, 1 week post-BOP and 1 month post-BOP), five animals were included. Three control animals were included for histo-morphometric analysis (an inter-animal comparison). All the experiments were performed as per the regulations and guidelines of the United States Department of Agriculture (USDA) and approved by the Institutional Animal Care and Use Committee of the University at Buffalo, NY. Chinchillas were unilaterally (ipsilateral – left side and contralateral – right side) exposed to a BOP (impulse) with its energy greatest below 10 kHz (see Fig 1 The frequency spectrum of acoustic energy) generated by a custom shock tube (Mark Cauble Precision Inc). As an attempt to mimic the real-world blast exposure, both the ipsilateral and contralateral ears were un-occluded conforming to the standard animal models for blast-induced neurotrauma.11 In this study, BOP was at ∼172 dB sound pressure level (SPL) (measured using a B & K Type 4938 microphone) with the pressure chamber at 23 psi with a duration of <2 ms (Fig. 2 The impulse waveform from our setup). The inter-aural BOP difference was at ∼6 dB SPL.

FIG. 1.

FIG. 1.

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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.

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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.

Auditory functional assays

DPOAEs were measured in a sound-attenuating room with insert earphones by playing two tones (at frequencies f1 and f2, f2/f1 = 1.2) from 0.5 to 10 kHz into the ear canal of anaesthetized chinchilla. DPOAEs were measured as SPL via an Etymōtic ER- 10B DPOAE probe system at distortion product frequency 2f1–f2. Pre- and 1 week (n = 12) and 1 month (n = 6) post-BOP DPOAE amplitudes were plotted as a function of 2F1–F2 frequencies at iso-intensity of 75 (F2)/65 (F1) dB SPL (Fig. 2). ABR input/output functions were obtained using tone burst frequencies of 0.5, 1, 2, 4, and 8 kHz in random order. Brain electrical activity was amplified 20,000 times (WPI model ISO-80, Sarasota, FL, USA), bandpass filtered from 0.3 to 3 kHz (Krohn-Hite model 3550 filter, Brockton, MA, USA), and digitally sampled (TDT instruments). ABR threshold was estimated using the cross-correlation method at each frequency and confirmed by visual inspection.

MR-DTI

DTI was acquired on chinchillas (n = 5) with a 200 mm diameter horizontal-bore 9.4 T magnet (Bruker Biospin, Biospec 94/20 USR) and a 440 mT/m (3440 T/m/s) imaging gradient system, which was operated with ParaVision (Bruker Biospin). We employed a cross-coil configuration with a four-channel phased-array receive coil (T10324V3; Bruker Biospin) placed over the head of the anesthetized chinchilla, and an 86 mm transmitter volume coil for excitation. Diffusion-weighted images were acquired using a 2D echo-planar imaging (EPI)-DTI with 30 diffusion directions (b = 1000 s/mm2) across the full sphere and four unweighted images, echo time 23 ms, repetition time 10 sec with a 0.3 mm gap and a 0.5 mm of slice thickness. A custom, semi-automated DTI processing pipeline was implemented to extract raw diffusion-weighted images and diffusion directions to calculate diffusion tensors in every voxel and convert them into DTI scalar metrics (Fig. 3). Diffusion-weighted images were corrected for eddy-current distortions, and diffusion tensor parameters were calculated on a voxel-by-voxel basis using DSI Studio.12 The DTI tensor values at CAN ROIs (as outlined in Fig. 4) were analyzed for three TPs: pre-BOP, and 1 week and 1 month after BOP by manually segmenting the ROIs using 3DSlicer. For each TP, a voxelwise t test was performed, and the five values (each voxel) from TP1 (pre-blast) are compared with the five values from TP2/TP3. Voxels with p < 0.05 were considered significant.

FIG. 3.

FIG. 3.

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(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.

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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.

Histological measures

Morphometric quantification of viable/dead neurons was performed in CAN ROIs of chinchillas (n = 3) at the 1-month post-BOP TP. The brains were fixed in 4% paraformaldehyde (PFA) and processed for routine paraffin processing. The sections (10 μm thick coronal slices of CN, IC and AC) were stained with CFV, H&E, and MT stains (Elsa Anton 1999). The stained sections were observed for morphological changes and photomicrographed under an upright Leica DM6B light microscope (Leica, Germany) at 400 × magnification. To quantify, the viable and dark stained neurons were observed with CFV stain and the apoptotic cells were identified using MT stain. With CFV (Fig. 5), and H&E (Fig. 6), Nissl-stained dark neurons and the intensely red-stained pyknotic nuclei reflected the cell shrinkage and condensation of both nucleus and cytoplasm.13–15 Cells that were undergoing apoptosis with apoptotic fragments were detected by differential red stain caused by fuchsin using MT stain (Fig. 7).16 For cell counting, the differential cell population in the CFV and MT-stained sections were counted manually from the average area (316.82 μm × 237.61 μm) of the photomicrograph which was considered as the field using “Cell counter” plugin in ImageJ software (National Institutes of Health [NIH], USA) in a blinded manner by multiple investigators. For analysis, viable/dark neurons and apoptotic cells were plotted in the ipsilateral and contralateral side for ROIs (CN, IC, and AC) based on features from CFV (rounded neurons/dark stained pyknotic neurons) and MT (dark red stained neurons) stains, respectively.

FIG. 5.

FIG. 5.

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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.

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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.

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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.

Statistical analysis

Statistical analysis was performed between groups across different TPs using GraphPad Prism (GraphPad Software, Inc). The histology, DTI, and ABR/DPOAE data were analyzed by two-way analysis of variance (ANOVA) repeated measures with a “post-hoc” Bonferroni multiple comparison test. For ABR and DPOAE, we compared the results in the same-animal at pre-BOP, 1 week post-BOP, and 1-month post-BOP TPs. Interaction between blast and frequency was analyzed. The values were represented as mean ± standard error of the mean (SEM). The values with p < 0.05 were considered as significant.

Results

Auditory functional assays

DPOAEs of individual chinchillas have been plotted for ipsilateral and contralateral ears (Fig. 2). In ipsilateral ear, DPOAE amplitudes at 1-week post-BOP were significantly decreased in all the frequencies when compared with pre-BOP DP amplitudes (p < 0.001). Overall, a single BOP significantly decreases DP amplitudes after 1 week but not after 1 month. In the contralateral ear, DP amplitudes 1 week post-BOP were significantly decreased when compared with pre-BOP at p < 0.01 for 4.3 kHz and at p < 0.001 for 3.7, 4.9, 5.5, 6.3, and 7.07 kHz. However, DP amplitudes were not decreased in 1-month post-BOP measurements when compared with pre-BOP except at 4.9 and 6.3 kHz (p < 0.05). Figure 8 shows ABR thresholds across pre-BOP, 1 week post-BOP and 1 month post-BOP frequencies, for both the ear ipsilateral and the ear contralateral to the BOP. The ABR thresholds of the ipsilateral ear were found to be increased significantly (p < 0.001) 1 week post-BOP at all frequencies when compared with pre-BOP ABR thresholds. Similarly, ABR thresholds of the contralateral ear were also found to be elevated significantly at all frequencies 0.5 and 2 (p < 0.01) and at 1 and 8 kHz (p > 0.001). The ABR thresholds of the ipsilateral ear at 1 month post-BOP were found to be elevated significantly at 0.5, 1, and 8 kHz (p < 0.001) as well as at 4 kHz (p < 0.01) when compared with pre-BOP ABR thresholds. At 1 month post-BOP, the ABR thresholds of the contralateral ear were elevated significantly only at 1, 2, and 4 kHz (p < 0.05) when compared with pre-BOP thresholds.

FIG. 8.

FIG. 8.

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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.

Voxel-based analysis of ROIs in DTI

AD was found to be significantly decreased in the ipsilateral CN at 1 month post-BOP (p < 0.05) when compared with the pre-BOP TP. Additionally, AD was significantly reduced in both the ipsilateral (p < 0.05) and contralateral (p < 0.001) CN at 1 month post-BOP when compared with the pre-BOP TP (Fig. 9-i). RD was significantly reduced (p < 0.05) in the contralateral CN at 1 week post-BOP when compared with the pre-BOP TP (Fig. 9-ii). The MD of the contralateral CN at 1 week was significantly reduced when compared with pre-BOP (Fig. 9-iii). FA was not significantly different between TPs (Fig. 9-iv). In addition, AD, RD, MD, and FA were not significantly different in CAN ROIs between the ipsilateral and contralateral ROIs. Overall, we found that there was no change in the tensor values in the AC and IC. However, in the CN, the AD was decreased significantly at 1 week and 1 month post-BOP. Similarly, RD was decreased at 1 week post-BOP and MD was found to be decreased in CN at 1 month post-BOP when compared with pre-BOP measures.

FIG. 9.

FIG. 9.

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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).

Histomorphometric analysis

Morphological changes were observed following CFV, H&E, and MT staining in control (n = 3) and 1 month post-BOP animals (n = 3). When compared with controls, viable cells were decreased significantly (p < 0.001) along with increased dying/apoptotic cells (p < 0.001) on both the ipsilateral and contralateral sides of AC and CN in BOP-exposed animals (Figs. 5 and 7). In the IC, when compared with controls, viable neurons were decreased significantly on the contralateral side, at p < 0.01, and dark neurons along with apoptotic cells were increased significantly at p < 0.001. When compared with controls, the reduction in viable neurons on the ipsilateral side of the IC was significant only at p < 0.05. Between the hemibrain of BOP-exposed animals, at the CN, viable cells were significantly decreased, with significantly increased apoptotic neurons (p < 0.05) in the ipsilateral side when compared with the contralateral side. Similarly, dark neuron counts were increased significantly at the ipsilateral side (p < 0.001) when compared with the contralateral side. At the contralateral IC, metrics such as those for viable cell reduction, an increase of apoptotic cells, and dead neuron counts were significant at p < 0.05, p < 0.01, and p < 0.001, respectively, when compared with the ipsilateral side. At the AC, although there was no significant difference between viable cell counts, degenerating cells and apoptotic cells were significantly elevated in the ipsilateral side when compared with the contralateral side. Compared with CFV and MT staining, a similar pattern of red dead neurons was observed in qualitative observations in the coronal slices of control and BOP-exposed chinchillas' CAN ROIs stained with H& E (Fig. 6).

Discussion

Among the neurological consequences, hearing loss and tinnitus top the disability claims of veterans and service members. Unfortunately, pre-clinical studies focusing on blast-induced pathology on the auditory periphery17–22 and central auditory neurodegeneration are limited.17,18,23-27 Chinchillas have long been used to study the effects of noise exposure,28 and extensive behavioral studies have been performed, including measures of frequency and intensity discrimination29,30 and localization.31 Chinchillas are a suitable model for auditory research following blast trauma14 because of their similarity with humans in terms of their frequency range of hearing.32,33 Unfortunately, few studies have investigated the effect of BOP on the auditory periphery and neurodegeneration of the central auditory pathway in chinchillas.4,5,34–38 Our discussion is limited to the studies on chinchillas.

Effect of single BOP on auditory functional integrity

Previous reports have demonstrated that the critical level of noise exposure to cause permanent threshold shift is ≥150 dB SPL.39 Following a single unilateral BOP at ∼172 dB SPL in the ipsilateral ear to the BOP (and ∼166 dB pSPL in the contralateral ear), the ABR threshold shifts have been increased 1 week post-BOP in the ipsilateral ear, which was found to be recovered at 1 month post-BOP . Greater than 30 dB and 10 dB (mean) threshold shifts across frequencies have been observed at 1 week and 1 month post-BOP, respectively. A similar pattern was observed in earlier work, in which chinchillas were exposed to repetitive impulse noise exposures (at 113 dB SPL).40 In our data, the same pattern was observed in DPOAE amplitude shifts at both post-BOP TPs. In our limited sample, we have not detected any DPs above our recording noise floor in side ipsilateral to BOP at 1 week post-BOP. A similar analysis of functional integrity in chinchilla was performed by Patterson and coworkers,41 Hickman and coworkers,4 Chen and coworkers,5 and Smith and coworkers.36 Our exposure of one blast at 172 dB SPL is similar to one of the exposures described in Hickman and coworkers.4 Our functional data indicate that damage to both the auditory periphery and neural generators of ABRs is more pronounced at week 1 post-BOP than at 1 month post-BOP.

Effect of single BOP on the microarchitecture of CAN

As DTI on blast-injured pre-clinical models indicates that DTI provides a valuable insight on BOP-induced pathobiology,42,43 we analyzed the tissue microarchitecture in chinchilla using DTI in the CN, IC, and AC. We observed no significant changes in diffusion properties of AC and IC pre- versus post-BOP. Importantly, however, we found that molecular diffusion rate along the axial direction (that refers axial diffusivityAD) was found to be decreased in the ipsilateral CN at 1 month post-BOP and in the contralateral CN at 1 week and 1 month post-BOP. Evidence indicates that only FA and RD are strongly correlated with electrophysiological markers,44 which explains the inconsistency between AD and ABR thresholds. In the present study, we restricted our DTI acquisition to ROIs and have not obtained the whole brain DTI. However, MD and AD data of CN at 1 month post-BOP correlate with the residual damage observed in the ABR and DPOAE shifts. The only other study27 that investigated the effect of BOP on ROIs (CN, IC, AC, medial geniculate bodies (MGB), amygdala, and corpus callosum) that included the auditory pathway was on rats using a single blast at 194 dB pSPL. This study showed abnormal AD of IC and MGB acutely, which returns back to pre-BOP level at 1 month post-BOP. However, our data in chinchilla showed no change in tensor values in IC. Further, our data indicated decreased AD in CN at 1 month post-BOP. The CN is the first auditory nucleus of the brain, receiving input from auditory nerve fibers. Our results are consistent with those of Budde and coworkers,45 who reported no significant difference between FA values of the cortex at 1month post-BOP.

Effect of single BOP on cellular changes in the CAN

Prior work on impulse noise on chinchillas' auditory periphery indicated cochlear ultrastructural changes in animals exhibiting post-traumatic stress (PTS),46 and the degree of damage was reflected in threshold elevation.47,48 Following blast exposure,4,21 differential ciliary changes have been observed in chinchillas' inner and outer hair cells.49 However, irrespective of species, the studies on the effect of BOP on the central auditory pathway is very limited despite the evidence on the effect of continuous noise exposure on chinchillas' central auditory neuraxis.50 In rats, following three blasts, progressive retrograde neurodegeneration and Tau accumulation was reported in the AC.51 However, following a single blast (197 dB SPL), astrocytosis and axonal degeneration were reported in AC 3 months post-bast. Recently, in chinchillas' AC, following blast, Smith and coworkers36 found an inverse relationship between the expression of a second messenger PI3K and hearing function. From our results of morphometric analysis of CAN ROIs, it is evident that a single unilateral BOP at 172 dB SPL reduced the counts of viable neurons and increased the dark neurons in ipsilateral CN, IC, and AC coronal slices. The degenerating neurons were eventually dying based on the evidence of increased apoptotic bodies in CAN ROIs. However, evidence of apoptotic bodies was more significant only in the CN and AC, not in the IC. Interestingly, the apoptotic bodies were increased more in the contralateral IC. We speculate that this might be for two reasons: that the IC is receiving inputs from both sides and that we had not occluded the contralateral ear.

Conclusion

Although we observed microarchitectural changes only at the level of the CN, the central cytoarchitecture is compromised at all levels. Despite the evidence of single unilateral BOP-induced pathology in chinchillas' central auditory neuraxis, further work is warranted in terms of peripheral anatomic measures (measurement of hair cell loss, synaptic changes, measures of stria vascularis integrity) and synaptic changes at the CN, IC, and AC. Continuing investigation of cellular and molecular changes in chinchilla's central auditory neuraxis could provide a platform to validate potential therapeutic approaches aimed at reducing blast-induced auditory neurodegeneration.

Acknowledgments

We thank Dr. Robert F. Burkard, University at Buffalo for helping us with impulse wave calibration and Dr. Michael G. Heinz and Dr. Satyabrata Parida, Purdue University, for helping with custom MATLAB scripts and technical inputs toward the non-invasive functional assays. We also thank members of UB Health Sciences Instrument Shops (Mr. John Franke, Mr. Michael Fletcher, and Mr. Brian Koyn).

Authors' Contributions

Kathiravan Kaliyappan performed blast-exposures, functional assays (ABR and DPOAE), histology, and histomorphometric analysis; Johan Nakuci completed the post-processing of DTI analysis and extracted the various tensor values and respective statistical analysis. Marilena Preda acquired the DTI for all TP. Ferdinand Schweser optimized the DTI acquisition setup and DTI acquisition parameters, devised the DTI data extraction pipeline and exported the DTI raw data for analysis, and provided training in 3DSlicer. Sarah Muldoon reviewed the pipeline of post-processing of DTI tensor data. Vijaya Prakash Krishnan Muthaiah conceptualized the study design, resources, and data acquisition, reviewed the data analysis of all parameters, ran statistical analysis, and prepared the manuscript.

Funding Information

This work was made possible with the help of seed funding provided to Vijaya Prakash Krishnan Muthaiah, by the General Grand Chapter Royal Arch Masons International through Emerging Research Grants-Hearing Health Foundation. Research reported in this publication was funded by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR001412. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Author Disclosure Statement

No competing financial interests exist.

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