Age-Related Inflammation and Oxidative Stress in the Cochlea Are Exacerbated by Long-Term, Short-Duration Noise Stimulation.

We have previously reported that young adult rats exposed to daily, short-duration noise for extended time periods, develop accelerated presbycusis starting at 6 months of age. Auditory aging is associated with progressive hearing loss, cell deterioration, dysregulation of the antioxidant defense system, and chronic inflammation, among others. To further characterize cellular and molecular mechanisms at the crossroads between noise and age-related hearing loss (ARHL), 3-month-old rats were exposed to a noise-accelerated presbycusis (NAP) protocol and tested at 6 and 16 months of age, using auditory brainstem responses, Real-Time Reverse Transcription-Quantitative PCR (RT-qPCR) and immunocytochemistry. Chronic noise-exposure leading to permanent auditory threshold shifts in 6-month-old rats, resulted in impaired sodium/potassium activity, degenerative changes in the lateral wall and spiral ganglion, increased lipid peroxidation, and sustained cochlear inflammation with advancing age. Additionally, at 6 months, noise-exposed rats showed significant increases in the gene expression of antioxidant enzymes (superoxide dismutase 1/2, glutathione peroxidase 1, and catalase) and inflammation-associated molecules [ionized calcium binding adaptor molecule 1, interleukin-1 beta (IL-1ß), and tumor necrosis factor-alpha]. The levels of IL-1ß were upregulated in the spiral ganglion and spiral ligament, particularly in type IV fibrocytes; these cells showed decreased levels of connective tissue growth factor and increased levels of 4-hydroxynonenal. These data provide functional, structural and molecular evidence that age-noise interaction contributes to exacerbating presbycusis in young rats by leading to progressive dysfunction and early degeneration of cochlear cells and structures. These findings contribute to a better understanding of NAP etiopathogenesis, which is essential as it affects the life quality of young adults worldwide.

Age-Related Hearing Loss Is Accelerated by Repeated Short-Duration Loud Sound Stimulation.

Both age-related hearing loss (ARHL) and noise-induced hearing loss (NIHL) may share pathophysiological mechanisms in that they are associated with excess free radical formation and cochlear blood flow reduction, leading to cochlear damage. Therefore, it is possible that short, but repeated exposures to relatively loud noise during extended time periods, like in leisure (i.e., musical devices and concerts) or occupational noise exposures, may add to cochlear aging mechanisms, having an impact on the onset and/or progression of ARHL. Consequently, the aim of the present study was to determine if repeated short-duration overexposure to a long-term noise could accelerate permanent auditory threshold shifts associated with auditory aging in an animal model of ARHL. Toward this goal, young adult, 3-month-old Wistar rats were divided into two groups: one exposed (E) and the other non-exposed (NE) to noise overstimulation. The stimulation protocol consisted of 1 h continuous white noise at 110 dB sound pressure level (SPL), 5 days a week, allowing 2 days for threshold recovery before initiating another stimulation round, until the animals reached an age of 18 months. Auditory brainstem response (ABR) recordings at 0.5, 1, 2, 4, 8, 16, and 32 kHz were performed at 3, 6, 12, and 18 months of age. The results demonstrate that in the E group there were significant increases in auditory thresholds at all tested frequencies starting already at 6 months of age, which extended at 12 and 18 months. However, in NE animals threshold shifts were not evident until 12 months, extending to 18 months of age. Threshold shifts observed in the E animals at 6 and 12 months were significantly larger than those observed in the NE group at the same ages. Threshold shifts at 6 and 12 months in E animals resembled those at 12 and 18 months in NE animals, respectively. This suggests that repeated noise overstimulation in short-duration episodes accelerates the time-course of hearing loss in this animal model of ARHL.

Validation of Reference Genes for RT-qPCR Analysis in Noise-Induced Hearing Loss: A Study in Wistar Rat.

The reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) requires adequate normalization in order to ensure accurate results. The use of reference genes is the most common method to normalize RT-qPCR assays; however, many studies have reported that the expression of frequently used reference genes is more variable than expected, depending on experimental conditions. Consequently, proper validation of the stability of reference genes is an essential step when performing new gene expression studies. Despite the fact that RT-qPCR has been widely used to elucidate molecular correlates of noise-induced hearing loss (NIHL), up to date there are no reports demonstrating validation of reference genes for the evaluation of changes in gene expression after NIHL. Therefore, in this study we evaluated the expression of some commonly used reference genes (Arbp, b-Act, b2m, CyA, Gapdh, Hprt1, Tbp, Tfrc and UbC) and examined their suitability as endogenous control genes for RT-qPCR analysis in the adult Wistar rat in response to NIHL. Four groups of rats were noise-exposed to generate permanent cochlear damage. Cochleae were collected at different time points after noise exposure and the expression level of candidate reference genes was evaluated by RT-qPCR using geNorm, NormFinder and BestKeeper software to determine expression stability. The three independent applications revealed Tbp as the most stably expressed reference gene. We also suggest a group of top-ranked reference genes that can be combined to obtain suitable reference gene pairs for the evaluation of the effects of noise on gene expression in the cochlea. These findings provide essential basis for further RT-qPCR analysis in studies of NIHL using Wistar rats as animal model.

Synergistic effects of free radical scavengers and cochlear vasodilators: a new otoprotective strategy for age-related hearing loss.

The growing increase in age-related hearing loss (ARHL), with its dramatic reduction in quality of life and significant increase in health care costs, is a catalyst to develop new therapeutic strategies to prevent or reduce this aging-associated condition. In this regard, there is extensive evidence that excessive free radical formation along with diminished cochlear blood flow are essential factors involved in mechanisms of other stress-related hearing loss, such as that associated with noise or ototoxic drug exposure. The emerging view is that both play key roles in ARHL pathogenesis. Therapeutic targeting of excessive free radical formation and cochlear blood flow regulation may be a useful strategy to prevent onset of ARHL. Supporting this idea, micronutrient-based therapies, in particular those combining antioxidants and vasodilators like magnesium (Mg(2+)), have proven effective in reducing the impact of noise and ototoxic drugs in the inner ear, therefore improving auditory function. In this review, the synergistic effects of combinations of antioxidant free radicals scavengers and cochlear vasodilators will be discussed as a feasible therapeutic approach for the treatment of ARHL.

Upregulation of insulin-like growth factor and interleukin 1ß occurs in neurons but not in glial cells in the cochlear nucleus following cochlear ablation.

One of the main mechanisms used by neurons and glial cells to promote repair following brain injury is to upregulate activity-dependent molecules such as insulin-like growth factor 1 (IGF-1) and interleukin-1ß (IL-1ß). In the auditory system, IGF-1 is crucial for restoring synaptic transmission following hearing loss; however, whether IL-1ß is also involved in this process is unknown. In this study, we evaluated the expression of IGF-1 and IL-1ß within neurons and glial cells of the ventral cochlear nucleus in adult rats at 1, 7, 15, and 30 days following bilateral cochlear ablation. After the lesion, significant increases in both the overall mean gray levels of IGF-1 immunostaining and the mean gray levels within cells of the cochlear nucleus were observed at 1, 7, and 15 days compared with control animals. The expression and distribution of IL-1ß in the ventral cochlear nucleus of ablated animals was temporally and spatially correlated with IGF-1. We also observed a lack of colocalization between IGF-1 and IL-1ß with either astrocytes or microglia at any of the time points following ablation. These results suggest that the upregulation of IGF-1 and IL-1ß levels within neurons-but not within glial cells-may reflect a plastic mechanism involved in repairing synaptic homeostasis of the overall cellular environment of the cochlear nucleus following bilateral cochlear ablation.