Noise-induced hearing loss: Neuropathic pain via Ntrk1 signaling

Abstract

Severe noise-induced damage to the inner ear leads to auditory nerve fiber degeneration thereby reducing the neural input to the cochlear nucleus (CN). Paradoxically, this leads to a significant increase in spontaneous activity in the CN which has been linked to tinnitus, hyperacusis and ear pain. The biological mechanisms that lead to an increased spontaneous activity are largely unknown, but could arise from changes in glutamatergic or GABAergic neurotransmission or neuroinflammation. To test this hypothesis, we unilaterally exposed rats for 2h to a 126dB SPL narrow band noise centered at 12kHz. Hearing loss measured by auditory brainstem responses exceeded 55dB from 6 to 32kHz. The mRNA from the exposed CN was harvested at 14 or 28days post-exposure and qRT-PCR analysis was performed on 168 genes involved in neural inflammation, neuropathic pain and glutamatergic or GABAergic neurotransmission. Expression levels of mRNA of Slc17a6 and Gabrg3, involved in excitation and inhibition respectively, were significantly increased at 28days post-exposure, suggesting a possible role in the CN spontaneous hyperactivity associated with tinnitus and hyperacusis. In the pain and inflammatory array, noise exposure upregulated mRNA expression levels of four pain/inflammatory genes, Tlr2, Oprd1, Kcnq3 and Ntrk1 and decreased mRNA expression levels of two more genes, Ccl12 and Il1β. Pain/inflammatory gene expression changes via Ntrk1 signaling may induce sterile inflammation, neuropathic pain, microglial activation and migration of nerve fibers from the trigeminal, cuneate and vestibular nuclei into the CN. These changes could contribute to somatic tinnitus, hyperacusis and otalgia.

Keywords: Cochlear nucleus; GABA; Gene array; Glutamate; Neuropathic pain; Noise exposure; Sterile inflammation.

Figure 1

Mean (+/− SEM) ABR thresholds in the right-exposed ear ranged from 90-100 decibels sound pressure level (dB SPL) between 6 and 32 kHz at 14 and 28 days post-exposure (open symbols; n=6). ABR thresholds in the left-plugged sham control ear were approximately 40 dB SPL (green, filled symbol, n=6). ABR thresholds measured in the anesthetized, but unexposed control animals (filled symbols) were approximately 40 dB at 14 days and 28 days post-anesthesia (n=6).

Figure 2

Gene array volcano plots for GABA and glutamate (upper half) and neuropathic pain and inflammatory (lower half) gene arrays. Each volcano plot shows the Log2 (Fold-Change) in gene expression on the X-axis versus Log10 (p-Value) of statistical significance on the Y-axis (mean of 4 replicates). Red dots indicate fold increases above 0.5 (increases >50%) while the blue dots indicate fold decreases below −0.5 (decreases >50%). Data points above the dotted horizontal line indicate statistically significant gene expression changes (p <0.05). Fig. 2 A and B show GABA and glutamate gene expression changes obtained at 14 days (A) and 28 days post-exposure (B). Fig. 2 C and D demonstrate neuropathic pain and inflammatory gene expression changes obtained at 14 days (C) and 28 days (D) post-exposure.

Figure 3

Western blots and histograms depict noise-induced changes in (A) Ntrk1, (B) Slc17a6 (C) Kcnq3 and (D) Gabrg3 protein expression at 2, 14 and 28 days post-exposure. The protein bands for Ntrk1, Slc17a6, Kcnq3 and Gabrg3 protein were normalized with the housekeeping protein actin. Ntrk1, Kcnq3 and Gabrg3 protein expression levels were significantly down-regulated relative to control (p < 0.001) at 2, 14 and 28 days post-exposure while Slc17a6 protein expression was significantly upregulated relative to control (p < 0.05) at 14 days post-exposure. Asterisks (*) denote significant differences between sham and noise exposure groups, while the pound symbol (#) indicates significant differences between recovery durations. ns = non-significant.

Figure 4

Immunolabeling of Gabrg3 in the ventral cochlear nucleus (VCN). Representative photomicrographs from an unexposed sham control rat at low (A) and high (C) magnifications and from a rat that recovered 28 days after noise at low (B) and high (D) magnifications. Low magnification images marked with a grey dotted border line in Fig. 4 A and B inserts show the overview of VCN (D-dorsal, L-lateral, GCL-Granule cell lamina, DCN Dorsal cochlear Nucelus) with a black star symbol inside the border. This star indicates the location of each high magnification image. These high magnification images show dark Gabrg3 puncta in and around the periphery of the soma and the surrounding neuropils.

Figure 5

Representative fluorescent photomicrographs exhibit Slc17a8 (A, D), Gabrg3 (B, E) immunolabeling and Slc17a8/Gabrg3 merged images (C, F) taken from the ventral cochlear nuclei (VCN) of an unexposed control rat (A-C) and of a 28 day post-exposure rat (D-F). In Fig. 5A & D, Slc17a8 (green) is heavily expressed in the soma (arrows) of neurons; while scattered puncta and patches are present in the surrounding neuropils. The overview of VCN anatomy is showed in Fig. 5 B & E inserts marked with a white dotted border line (D-dorsal, M-Medial). A star symbol in the insert localizes where the high magnification image is obtained. Slc17a8 labeling in 28 day post-exposure rat (D) is similar to that in control rat (A). In Fig. 5B & E, Gabrg3 (red) immunolabels surround numerous soma of neurons (arrows) and also are present in patches and puncta in the surrounding neuropils. In Fig. 5C & F, a merge of Slc17a8 and Gabrg3 labels demonstrates that Gabrg3 co-localizes the perimeter of Slc17a8-positive neurons in the cochlear nucleus of both control and noise-exposed rats at 28 days post-exposure (as indicated by red-filled arrows). Inserts in Fig. 5C and F depict higher magnification images of Gabrg3 engulfing most, if not all, of the Slc17a8-positive neurons in both sham (C) and 28 days recovered rat VCN. Scale bar = 50 μm.