Pioglitazone Represents an Effective Therapeutic Target in Preventing Oxidative/Inflammatory Cochlear Damage Induced by Noise Exposure

Abstract

Recent progress in hearing loss research has provided strong evidence for the imbalance of cellular redox status and inflammation as common predominant mechanisms of damage affecting the organ of Corti including noise induced hearing loss. The discovery of a protective molecule acting on both mechanisms is challenging. The thiazolidinediones, a class of antidiabetic drugs including pioglitazone and rosiglitazone, have demonstrated diverse pleiotrophic effects in many tissues where they exhibit anti-inflammatory, anti-proliferative, tissue protective effects and regulators of redox balance acting as agonist of peroxisome proliferator-activated receptors (PPARs). They are members of the family of ligand regulated nuclear hormone receptors that are also expressed in several cochlear cell types, including the outer hair cells. In this study, we investigated the protective capacity of pioglitazone in a model of noise-induced hearing loss in Wistar rats and the molecular mechanisms underlying this protective effects. Specifically, we employed a formulation of pioglitazone in a biocompatible thermogel providing rapid, uniform and sustained inner ear drug delivery via transtympanic injection. Following noise exposure (120 dB, 10 kHz, 1 h), different time schedules of treatment were employed: we explored the efficacy of pioglitazone given immediately (1 h) or at delayed time points (24 and 48 h) after noise exposure and the time course and extent of hearing recovery were assessed. We found that pioglitazone was able to protect auditory function at the mid-high frequencies and to limit cell death in the cochlear basal/middle turn, damaged by noise exposure. Immunofluorescence and western blot analysis provided evidence that pioglitazone mediates both anti-inflammatory and anti-oxidant effects by decreasing NF-κB and IL-1β expression in the cochlea and opposing the oxidative damage induced by noise insult. These results suggest that intratympanic pioglitazone can be considered a valid therapeutic strategy for attenuating noise-induced hearing loss and cochlear damage, reducing inflammatory signaling and restoring cochlear redox balance.

Keywords: PPAR agonist; acoustic trauma; antinflammatory; antioxidant; audiology; personalized medicine.

FIGURE 1

Pioglitazone protects against noise-induced hearing loss. (A–F) Graphs show mean threshold shift values (means ± SEM) for mid frequencies (12–16 kHz, A,C,E) and high frequencies (20–24–32 kHz, B,D,F) measured 1, 3, 7, 14 and 21 days after vehicle (black squares; n = 24) or pioglitazone injection (gray diamonds; n = 24) in animals exposed to noise and treated 1 h (A,B), 24 h (C,D), or 48 h (E,F) after the acoustic trauma. (G–I) Graphs show threshold values (means ± SEM) across all frequencies analyzed at day 21 in animals exposed to noise and treated with pioglitazone or vehicle at different time points. Baseline values refer to auditory thresholds estimated prior to pioglitazone or vehicle injection in each condition. Pioglitazone administered 1 h after acoustic trauma provides the major protection, attenuating threshold shift by about 15 dB for mid frequencies and 20 dB for high frequencies. Notably, low frequency (6 kHz) was less affected by acoustic trauma, consistent with our noise exposure protocol (pure tone centered to 10 kHz). Delayed administration (24 and 48 h post noise exposure) shows significant but slightly minor protection (about 10-15 dB). A-F: asterisks indicate significant differences between groups (^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001). (G–I) Asterisks refer to significant differences between Noise + Pio and Noise + Vehicle groups (^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001).

FIGURE 2

Morphological evaluation and hair cell count. (A–F) Representative images of surface preparation of the organ of Corti showing F-actin distribution in the middle-basal cochlear turn. A typical distribution of three rows of outer hair cells (OHCs) and one row of inner hair cells (IHCs) is shown in (A). Noise exposure (B) caused OHC loss (dark spots, indicated with arrows). Pioglitazone significantly reduced OHC death (D–F) with respect to Noise-vehicle condition (C), specifically if administered 1 h after noise insult (D). Scale bar: 20 μm. (G–I) Cochleograms (means ± SEM) showing percentage of OHC survival in the three different administration schedules. Noise induced about 40% and 20% of cell death in the middle and basal turns respectively. Pioglitazone administration attenuates cell death of about 20–25%. Asterisks indicate significant differences between groups (^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; n = 6 animals/group).

FIGURE 3

Early pioglitazone administration reduces superoxide amount in the cochlea. (A–F) Representative images of cochlear cryo-sections (middle-basal turns) stained with DHE (red fluorescence). Insets of the organ of Corti are shown in (C,D). Superoxide fluorescence was faint in the cytoplasm of control cochleae (A). Noise exposure increased superoxide amount, mainly in spiral ganglion neurons, organ of Corti and stria vascularis (B). No difference in superoxide amount was found between Noise and Noise-vehicle conditions (B,C). When administered 1 h after noise exposure, pioglitazone significantly decreased superoxide expression in the main cochlear structures (D). Administration of either 24 h (E) and 48 h (F) post-noise also reduced superoxide but to a lesser extent as compared to earlier administration. Scale bar: 100 μm. (G) Histograms (means ± SEM), related to Noise-Pio and Noise + vehicle groups, show quantification of fluorescence intensity (A.U., arbitrary units) in the principal cochlear structures: spiral ganglion neurons (SGNs), organ of Corti (oC) and stria vascularis (StV). Asterisks indicate significant differences between groups (^∗p < 0.05; n = 12 slices selected randomly from 6 animals for each experimental group).

FIGURE 4

Pioglitazone reduces lipid peroxidation when administered 1 h after noise exposure. (A–F) Representative images of cochlear cryo-sections (middle-basal turns) stained with a marker of lipid peroxidation (8-Isoprostane, green fluorescence) and stained with DAPI (blue fluorescence). Insets of high magnification of the organ of Corti are shown in (C,D). Noise exposure induces an increase of lipid peroxidation in all cochlear structures (B), with no difference with respect to animals exposed to noise and treated with vehicle (C). Lipid peroxidation was absent in the cytoplasm of control cochleae (A). Noise exposure increased 8-Isoprostane amount, mainly in spiral ganglion neurons, organ of Corti and specifically stria vascularis (see arrow in B). No difference was found between Noise and Noise-vehicle conditions (C). When administered 1 h after noise exposure, pioglitazone significantly decreased 8-Isoprostane expression (D). Administration 24 h (E) and 48 h (F) post noise showed small but detectable effect in counteracting lipid peroxidation. Scale bar: 100 μm. (G) Histograms (means ± SEM) show quantification of fluorescence intensity (A.U., arbitrary units) as described in Figure 3. SGNs, spiral ganglion neurons; oC, organ of Corti; StV, stria vascularis. Asterisks indicate significant differences between groups (^∗p < 0.05; ^∗∗p < 0.01; n = 12 slices selected randomly from 6 animals for each experimental group).

FIGURE 5

Pioglitazone reduces cochlear expression of NF-κB. (A–F) pNF-κB (red fluorescence) and DAPI staining (blue fluorescence) in cochlear cryo-sections (middle-basal turns) of all experimental groups. Insets of high magnification of the organ of Corti are shown in (C,D). NF-κB expression increased after noise exposure in all cochlear structures (B) with no differences between Noise and Noise-vehicle groups (C). Local delivery of pioglitazone attenuated NF-κB phosphorylation when administered 1 h post noise (D) as well as when administered either 24 and 48 h post noise (E,F). (G) Histograms (means ± SEM) show quantification of fluorescence intensity (A.U., arbitrary units) as described in Figure 3. SGNs, spiral ganglion neurons; oC, organ of Corti; StV, stria vascularis. Asterisks indicate significant differences between groups (^∗p < 0.05; ^∗∗p < 0.01; n = 12 slices selected randomly from 6 animals for each experimental group).

FIGURE 6

Pioglitazone counteracts the increase of IL-1β expression. (A–F) Representative images of IL-1β expression (green fluorescence) in cochlear cryo-sections (middle-basal turns) stained with DAPI (blue fluorescence). Insets of high magnification of the organ of Corti are shown (C,D). The IL-1β expression increase observed after noise exposure (B) was significantly reduced after pioglitazone treatment (D–F). No difference was found between Noise and Noise-vehicle groups (C). Scale bar: 100 μm. (G) Histograms (means ± SEM) show quantification of fluorescence intensity (A.U., arbitrary units) as described in Figure 3. SGNs, spiral ganglion neurons; oC, organ of Corti; StV, stria vascularis. Asterisks indicate significant differences between groups (^∗p < 0.05; ^∗∗p < 0.01; n = 12 slices selected randomly from 6 animals for each experimental group).

FIGURE 7

Pioglitazone targets the oxidative/inflammatory interplay in cochlear damage. (A,B) Graphs show the shift/decrease of fluorescence intensity (measured by comparing Noise-vehicle vs. Noise-Pio conditions) for the different immunofluorescence analysis (8-Isoprostane and NF-κB), at the different drug administration schedules (1, 24 or 48 h post noise exposure). Data are expressed as means ± SEM (A.U., arbitrary units). (C) Representative immuno-reactive bands for pNF-κB and IL-1β in controls and animals exposed to noise and treated with pioglitazone (N + P; n = 6) or vehicle (N + V; n = 6) at the different time points (1, 24, or 48 h after noise exposure) 7 days after administration. Reduction of both NF-κB and IL1-β was clearly detected 7 days after drug treatment.