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. 2020 Apr 13;21(8):2692.
doi: 10.3390/ijms21082692.

New Insights on the Effect of TNF Alpha Blockade by Gene Silencing in Noise-Induced Hearing Loss

Janaína C Rodrigues  1   2 , André L L Bachi  3   4   5 , Gleiciele A V Silva  2 , Marcelo Rossi  3 , Jonatas B do Amaral  3 , Karina Lezirovitz  1   2 , Rubens de Brito  1
Affiliations

New Insights on the Effect of TNF Alpha Blockade by Gene Silencing in Noise-Induced Hearing Loss

Janaína C Rodrigues et al. Int J Mol Sci. .

Abstract

Noise exposure represents the second most common cause of acquired sensorineural hearing loss and we observed that tumor necrosis factor α (TNFα) was involved in this context. The effect of Tnfα gene silencing on the expression profile related to the TNFα metabolic pathway in an experimental model of noise-induced hearing loss had not previously been studied.

Methods: Single ears of Wistar rats were pretreated with Tnfα small interfering RNA (siRNA) by trans-tympanic administration 24 h before they were exposed to white noise (120 dBSPL for three hours). After 24 h of noise exposure, we analyzed the electrophysiological threshold and the amplitude of waves I, II, III, and IV in the auditory brain response click. In addition, qRT-PCR was performed to evaluate the TNFα metabolic pathway in the ears submitted or not to gene silencing.

Results: Preservation of the electrophysiological threshold and the amplitude of waves was observed in the ears submitted to gene silencing compared to the ears not treated. Increased anti-apoptotic gene expression and decreased pro-apoptotic gene expression were found in the treated ears.

Conclusion: Our results allow us to suggest that the blockade of TNFα by gene silencing was useful to prevent noise-induced hearing loss.

Keywords: TNFα metabolic pathway; apoptosis; auditory brain response; cochlea; electrophysiological threshold; in vivo siRNA administration; synaptopathy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Fluorescence analysis of tumor necrosis factor α (TNFα) (green-labeled) in glial fibroblast cells treated with scramble small interfering RNA (siRNA) (negative control-A) or Tnfα siRNA (B). Shown in (C) is the silencing rate of the fluorescence intensity between the scramble siRNA and Tnfα siRNA (black bar, 96%). In (D) is the positive control of the delivery agent (red-labeled). The fluorescence of nine sites per well, with a total of three wells per treatment, was analyzed by MetaXpress software. Statistical differences in the values of TNFα labeling (A and B) were obtained using Student’s t-test, with the Bonferroni post-hoc test, at a significance level of 5% (p < 0 05).
Figure 2
Figure 2
A heat map showing the comparisons of differential gene expression of the TNFα metabolic pathway in the cochleae of rats previously submitted (n = 20) or not submitted (n = 20) to Tnfα siRNA administration and after noise exposure. According to the fold-change found by the qRT-PCR analysis, the “red” color is used to indicate the highest gene expression (up-regulated genes), whereas the “green” color is used to indicate the lowest gene expression (down-regulated genes). In the “gray” color are presented the gene expression values (or transcript values) that were poorly evaluated due to insufficient resolution or image noise, which, in a general way, are named as “missing values”.
Figure 3
Figure 3
Fold change of the main apoptotic genes that were up and down-regulated in the cochleae of rats previously submitted (S, n = 20) or not submitted (NS, scramble, n = 20) to TNFα blockade by gene silencing before and after noise exposure.
Figure 4
Figure 4
Representative analysis of the electrophysiological threshold shift (in dBSPL) in the cochleae of rats previously submitted (S, n = 20) or not submitted (NS, scramble, n = 20) to TNFα blockade by gene silencing before and after noise exposure. Narrow represents wave II, which was used to determine the threshold shift.
Figure 5
Figure 5
Analysis of the electrophysiological threshold shift (in dBSPL) in the cochleae of rats previously submitted (S, n = 20) or not submitted (NS, scramble, n = 20) to TNFα blockade by gene silencing before and after noise exposure. Differences in the electrophysiological threshold shift were evaluated by Student’s t-test with Bonferroni’s post-hoc test. The significance level was set as 5%. **** p < 0.0001.
Figure 6
Figure 6
Auditory brainstem response (ABR), evaluated by waves amplitude I, II, III, and IV (represented by bioelectric signals set at microvolts, μV) at 90 dBSPL (A) and at 80 dBSPL (B) in ears previously submitted (S, n = 20) or not submitted (NS, scramble, n = 20) to TNFα blockade by gene silencing and after to noise exposure. Differences in the wave’s amplitude were evaluated by Student’s t-test with Bonferroni´s post-hoc test. The significance level was set as 5%. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
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