Percent of outer hair cell (OHC) loss as a function of percent distance (and frequency-specific tonotopic position) from the OC apex in untreated and HPN-07-treated chinchilla at 3 days (d), 10 d, 21 d, and 180 d after noise exposure.

<p>The data are plotted as mean ± SEM and “n” represents the number of cochleae. Vertical lines at right of figures show a significant difference of mean values for noise only "Control" and Treated" groups as determined by two- way ANOVA followed by Bonferroni post-tests: *, <i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. Shaded area demarcates the range of noise exposure centered at 4 kHz. Significantly less OHC loss was observed in the basal turn of the OC in the HPN-07 "Treated" group (<i>p</i> < 0.05, 0.01 or 0.001).</p

Compound action potential (CAP) and cochlear microphonic (CM) threshold change as a function of test frequency over different time groups plotted in the same format as Fig 1.

<p>Each sampling group contained readings from six animals. The results show a decrease in both CAP and CM thresholds for HPN-07 treated animals relative to control animals beginning at 10 days after blast exposure. Significant differences were detected by two-way ANOVA analysis. The symbols of *; **; *** indicate significance levels of <i>p</i> < 0.05, 0.01 and 0.001, respectively.</p

HPN-07, a free radical spin trapping agent, protects against functional, cellular and electrophysiological changes in the cochlea induced by acute acoustic trauma - Fig 8

<p>Representative photomicrographs of cochlear hair cells after phalloidin staining at about 70% of the distance from the apex in normal (naïve) (A), and control (untreated) (B) and treated chinchilla at 21 days after the blast exposure. Arrows show examples of missing outer hair cells (OHC) that have been replaced by scars. Scale bar = 50 μm.</p

Effects of Antioxidant Treatment on Blast-Induced Brain Injury

<div><p>Blast-induced traumatic brain injury has dramatically increased in combat troops in today’s military operations. We previously reported that antioxidant treatment can provide protection to the peripheral auditory end organ, the cochlea. In the present study, we examined biomarker expression in the brains of rats at different time points (3 hours to 21 days) after three successive 14 psi blast overpressure exposures to evaluate antioxidant treatment effects on blast-induced brain injury. Rats in the treatment groups received a combination of antioxidants (2,4-disulfonyl α-phenyl tertiary butyl nitrone and N-acetylcysteine) one hour after blast exposure and then twice a day for the following two days. The biomarkers examined included an oxidative stress marker (4-hydroxy-2-nonenal, 4-HNE), an immediate early gene (c-fos), a neural injury marker (glial fibrillary acidic protein, GFAP) and two axonal injury markers [amyloid beta (A4) precursor protein, APP, and 68 kDa neurofilament, NF-68]. The results demonstrate that blast exposure induced or up-regulated the following: 4-HNE production in the dorsal hippocampus commissure and the forceps major corpus callosum near the lateral ventricle; c-fos and GFAP expression in most regions of the brain, including the retrosplenial cortex, the hippocampus, the cochlear nucleus, and the inferior colliculus; and NF-68 and APP expression in the hippocampus, the auditory cortex, and the medial geniculate nucleus (MGN). Antioxidant treatment reduced the following: 4-HNE in the hippocampus and the forceps major corpus callosum, c-fos expression in the retrosplenial cortex, GFAP expression in the dorsal cochlear nucleus (DCN), and APP and NF-68 expression in the hippocampus, auditory cortex, and MGN. This preliminary study indicates that antioxidant treatment may provide therapeutic protection to the central auditory pathway (the DCN and MGN) and the non-auditory central nervous system (hippocampus and retrosplenial cortex), suggesting that these compounds have the potential to simultaneously treat blast-induced injuries in the brain and auditory system.</p> </div

Connexin 26 relative fluorescence intensity in the OC was measured and statistically analyzed.

<p>Both noise exposed treated and control (untreated) groups showed a similar significant decrease in Cx-26 expression at 21 days and 6 months after noise exposure compared to the normal (naïve) animals (<i>p</i> < 0.05).</p

Bivariate analysis of the relationship between CM thresholds and EP for noise-exposed chinchilla in the HPN-07-treated and untreated groups (control).

<p>Data for low frequency (LF = 0.5 and 2 kHz); and high frequency (HF = 2, 4, 6, 8) kHz) were analyzed separately. Linear correlation analysis detected a correlation between EP and CM the in both the LF (r = 0.623, n = 24;) and HF (r = 0.676, n = 24) groups that were treated with HPN-07 following noise exposure but no correlation was detected in the untreated noise-exposed (n = 24) group.</p

Representative confocal images of connexin 26 (red) and 30 (green) labeling collected from the spiral ligament.

<p>Nuclei of cells were stained by DAPI (blue). Merged images are shown in the column 3 (A3-I3, merged). Positive Connexin 26 labeling (red) is observed in fibrocytes in the spiral ligament of normal (naïve) controls (A1). Decreased connexin 26 labeling (column 1) is observed in the spiral ligament of chinchilla all time points after noise exposure (B-H) except in the treated group at 6 months after noise exposure(I). No or very low CX-30 labeling is observed in fibrocytes of the spiral ligament of chinchilla while the root cells show positive Cx-30 labeling (A2-I2). Scale bar = 20 μm in I3 for A1-I3.</p

Examples of APP immunolabeling in the hippocampus of the NC (A), 24H-B (B) and 24H-B/T (C) groups.

<p>No positive APP staining was observed in the hippocampus of normal controls (A). Strong positive APP labeling was observed in the hippocampus of the 24H-B group (arrows in B). Decreased APP expression was observed in the hippocampus of the 24H-B/T group relative to the 24H-B group (arrows in C). APP-positive labeling in the hippocampus was quantified and statistically analyzed (D). Two to three hippocampal sections from each rat brain (6 rats in each group) were used in these analyses. Significantly increased APP expression was observed in the hippocampus of the 24H-B group compared to the NC group (<i>p</i> < 0.001). An antioxidant treatment effect was found at 24 hours after blast exposure (<i>p</i> < 0.001), however no significant difference was observed between the treated and untreated groups 7 days after blast exposure (7D-B v.s. 7D-B/T, all <i>p</i> > 0.05). Error bars represent standard error of the means. Scale bar = 10 µm in C for A-C. *** indicate <i>p</i> < 0.001.</p

Representative confocal images of connexin 26 (Cx-26) and 30 (Cx-30) labeling in the OCs.

<p>Positive Cx-26 labeling (red) is observed in supporting cells in the OC of normal (naïve) animals (A1). Positive CX-30 labeling (green) is observed in all cells in the OC of normal animals (A2). Merged images are shown in column 3. Decreased expression of Cx-26 is observed in the OCs of control (noise exposed, untreated) or treated groups (B1-E1). The noise exposure did not change the expression of Cx-30 in the OCs all time points after noise exposure (B2-E2). Scale bar = 20 μm in E3 for A1-E3.</p

Bivariate analysis of the relationship between click-evoked CAP thresholds and EP for normal and noise-exposed chinchilla that were either treated with HPN-07 or untreated (control).

<p>Noise exposure resulted in a persistent disruption of the normal close correlation between EP and CAP values whereas in the HPN-07 treated animals a linear relationship (r = 0.656, n = 24, p = 0.005) was found after noise exposure between the two measurements with a gradual return to normal values.</p