Temporal Changes in Functional and Structural Neuronal Activities in Auditory System in Non-Severe Blast-Induced Tinnitus

Abstract

Background and Objectives: Epidemiological data indicate that blast exposure is the most common morbidity responsible for mild TBI among Service Members (SMs) during recent military operations. Blast-induced tinnitus is a comorbidity frequently reported by veterans, and despite its wide prevalence, it is also one of the least understood. Tinnitus arising from blast exposure is usually associated with direct structural damage that results in a conductive and sensorineural impairment in the auditory system. Tinnitus is also believed to be initiated by abnormal neuronal activities and temporal changes in neuroplasticity. Clinically, it is observed that tinnitus is frequently accompanied by sleep disruption as well as increased anxiety. In this study, we elucidated some of the mechanistic aspects of sensorineural injury caused by exposure to both shock waves and impulsive noise. The isolated conductive auditory damage hypothesis was minimized by employing an animal model wherein both ears were protected. Materials and Methods: After the exposure, the animals' hearing circuitry status was evaluated via acoustic startle response (ASR) to distinguish between hearing loss and tinnitus. We also compared the blast-induced tinnitus against the well-established sodium salicylate-induced tinnitus model as the positive control. The state of the sensorineural auditory system was evaluated by auditory brainstem response (ABR), and this test helped examine the neuronal circuits between the cochlea and inferior colliculus. We then further evaluated the role of the excitatory and inhibitory neurotransmitter receptors and neuronal synapses in the auditory cortex (AC) injury after blast exposure. Results: We observed sustained elevated ABR thresholds in animals exposed to blast shock waves, while only transient ABR threshold shifts were observed in the impulsive noise group solely at the acute time point. These changes were in concert with the increased expression of ribbon synapses, which is suggestive of neuroinflammation and cellular energy metabolic disorder. It was also found that the onset of tinnitus was accompanied by anxiety, depression-like symptoms, and altered sleep patterns. By comparing the effects of shock wave exposure and impulsive noise exposure, we unveiled that the shock wave exerted more significant effects on tinnitus induction and sensorineural impairments when compared to impulsive noise. Conclusions: In this study, we systematically studied the auditory system structural and functional changes after blast injury, providing more significant insights into the pathophysiology of blast-induced tinnitus.

Keywords: blast overpressure; hearing loss; impulsive noise; tinnitus.

Figure 1

The results of acoustic startle response and PPI evaluation in: (A) in sham, (B) BLAST, and (C) SSIT cohorts. No statistically significant changes in GAP index nor PPI were noted for sham cohort (A). In the BLAST group, only changes in the GAP index on Day 1 and Day 28 were obvious (B). A significant change in the GAP index metric was also noted for the SSIT cohort on Day 7 and Day 28, but only at the 12–14 kHz band, not the broadband noise (2–30 kHz, (C)). OL, outliners. Repeated measure ANOVA was used for the statistical analysis of behavioral tests, with the following significance thresholds: * p < 0.05, and *** p < 0.0005.

Figure 1

The results of acoustic startle response and PPI evaluation in: (A) in sham, (B) BLAST, and (C) SSIT cohorts. No statistically significant changes in GAP index nor PPI were noted for sham cohort (A). In the BLAST group, only changes in the GAP index on Day 1 and Day 28 were obvious (B). A significant change in the GAP index metric was also noted for the SSIT cohort on Day 7 and Day 28, but only at the 12–14 kHz band, not the broadband noise (2–30 kHz, (C)). OL, outliners. Repeated measure ANOVA was used for the statistical analysis of behavioral tests, with the following significance thresholds: * p < 0.05, and *** p < 0.0005.

Figure 2

The results of acoustic startle response GAP (A–C) and PPI (D–F) evaluation in SSIT (A,D), sham (B,E), and BLAST (C,F) cohorts. The presented time-series data are average responses with subtracted baseline prescreening values. Statistical significance is indicated with asterisk (*, p < 0.05). The negative values indicate the decreasing response compared to baseline. Large inhibitory trend is evident in high-frequency bands in animals exposed to a single blast in both tests. This effect is relatively weak, and most of the time-frequency band groups are above the statistical significance threshold.

Figure 3

The auditory brainstem response thresholds after: (A). noise exposure (sham), (B). 180 kPa blast, and (C). continuous sodium salicylate injection. Both cohorts of animals subjected to noise and blast exposure had their ears protected by earplugs to avoid tympanic membrane rupture. Repeated measure ANOVA was used for the statistical analysis of behavioral tests, with the following significance thresholds: * p < 0.05, ** p < 0.005, and *** p < 0.0005.

Figure 3

The auditory brainstem response thresholds after: (A). noise exposure (sham), (B). 180 kPa blast, and (C). continuous sodium salicylate injection. Both cohorts of animals subjected to noise and blast exposure had their ears protected by earplugs to avoid tympanic membrane rupture. Repeated measure ANOVA was used for the statistical analysis of behavioral tests, with the following significance thresholds: * p < 0.05, ** p < 0.005, and *** p < 0.0005.

Figure 4

The auditory brainstem response thresholds after: (A). noise exposure (sham), (B). 180 kPa blast, and (C). Continuous sodium salicylate injection. The heatmap of differential ABR threshold values (treatment group data with subtracted baseline data, i.e., those taken before the treatment) are presented. Both cohorts of animals subjected to noise and blast exposure had their ears protected by earplugs to avoid tympanic membrane rupture.

Figure 5

CtBP2 immunohistochemistry staining images and normalized CtBP2 expression in auditory cortex according to immunofluorescence intensity. CtBP, Carboxyl terminal binding protein. The yellow arrows indicate the samples of the positive stains(the cells number are not counted instead the normalized fluorescence intensity is quantified and shown in the graph. Repeated measure ANOVA was used for the statistical analysis of behavioral tests, with the following significance thresholds: *** p < 0.0005.

Figure 6

GABAAR immunohistochemistry staining images and normalized GABAAR expression in auditory cortex according to immunofluorescence intensity. CtBP, Carboxyl terminal binding protein. The yellow arrows indicate the samples of the positive stains(the cells number are not counted instead the normalized fluorescence intensity is quantified and shown in the graph. Repeated measure ANOVA was used for the statistical analysis of behavioral tests, with the following significance thresholds: *** p < 0.0005.

Figure 7

NMDAR1 immunohistochemistry staining images and normalized NMDAR1 expression in auditory cortex according to immunofluorescence intensity. CtBP, Carboxyl terminal binding protein. The yellow arrows indicate the samples of the positive stains(the cells number are not counted instead the normalized fluorescence intensity is quantified and shown in the graph. Repeated measure ANOVA was used for the statistical analysis of behavioral tests, with the following significance thresholds: *** p < 0.0005.

Figure 8

Summary of EPM after sodium salicylate injection. (A) Closed arm distance; (B) Closed arm time; (C) Open arm distance; and (D) Open arm time. Repeated measure ANOVA was used for the statistical analysis of behavioral tests, with the following significance thresholds: * p < 0.05, ** p < 0.005, and *** p < 0.0005.

Figure 8

Summary of EPM after sodium salicylate injection. (A) Closed arm distance; (B) Closed arm time; (C) Open arm distance; and (D) Open arm time. Repeated measure ANOVA was used for the statistical analysis of behavioral tests, with the following significance thresholds: * p < 0.05, ** p < 0.005, and *** p < 0.0005.

Figure 9

Summary of EPM among sham and blast conditions: significant increase of the closed arm traveled distance in the BLAST group compared with sham group. (A) Closed arm distance; (B) Closed arm time; (C) Open arm distance; and (D) Open arm time. Repeated measure ANOVA was used for the statistical analysis of behavioral tests, with the following significance thresholds: * p < 0.05.

Figure 10

Summary of total sleep percentage (A), sleep percentage in the dark (B) and in the light (C) for three test groups used in the study. Repeated measure ANOVA was used for the statistical analysis of behavioral tests, with the following significance thresholds: * p < 0.05.