Effects of octreotide on SGNs <i>in vitro</i>.

<p>SGNs were treated with increasing concentrations of octreotide (1 µM, 10 µM and 20 µM, respectively) and compared to controls without octreotide treatment. Average number of SG neurites and average lenth of SG neurites observed from SG explants were analyzed. Representative SG explants stained with anti-200-kDa neurofilament antibody for each experimental condition are shown. Scale bar  = 300 µm.</p

Quantitative analysis of surviving HCs under different experimental conditions in the basal and middle cochlear turns.

<p>HC survival was significantly higher in groups treated with octreotide and gentamicin compared to with gentamicin treatment alone (<i>p</i><0.01 octreotide 1 µM, <i>p</i><0.01 octreotide 5 µM). No toxic effects were observed in OCs incubated with octreotide without gentamicin or with culture medium only (control). Asterisks indicate significant difference from treatment with gentamicin only (p<0.01 for all conditions). Data are expressed as the mean number of surviving HCs per 20 inner HCs. Vertical lines represent one standard deviation. <i>n</i> = 6 for each experimental condition.</p

Cochlear gene expression of SST receptor-2 in the OC of postnatal day (P)14 and P21 wild-type (WT) and SST receptor-1 knockout (KO) mice.

<p>The relative distribution of SST receptor-2 mRNA expression in OC tissue from wild-type (WT) and SST receptor-1 KO of different postnatal ages was quantified by real-time PCR. GAPDH was used as an endogenous control. Gene expression levels are expressed as the mean (± one standard deviation) fold increase compared to the values obtained in OC explants from P14 and P21 WT and SST receptor-1 knockout mice. Data were obtained from five independent experiments, per condition in each experiment 5 OCs were used. **<i>p</i><0.001 using Student's <i>t</i>-test.</p

Effects of octreotide and the Akt inhibitor SH-6 on gentamicin-induced HC damage.

<p>Three orderly rows of outer HCs (OHC) and a single row of inner HCs (IHC) were observed in OCs exposed to 10 µM SH-6. Comparatively, OCs cultured with gentamicin showed significant loss of HCs. Addition of octreotide to gentamicin-treated OCs resulted in significantly decreased HC loss compared to in those treated with gentamicin only. However, addition of SH-6 and octreotide did not result in decreased hair cell loss. Scale bar  = 20 µm.</p

Effects of octreotide on gentamicin-induced HC damage.

<p>Three orderly rows of outer HCs (OHC) and a single row of inner HCs (IHC) were observed in control OCs and in OCs exposed to 5 µM octreotide without gentamicin. Comparatively, OCs cultured with gentamicin showed significant loss of HCs. Addition of increasing concentrations of octreotide to gentamicin-treated OCs resulted in significantly decreased HC loss compared to in those treated only with gentamicin. Scale bar  = 20 µm.</p

Average number of SG neurites observed from SG explants under the different experimental conditions.

<p>The number of neurites observed on control are compared to those seen with three different levels of octreotide (Oct). Lines represent one standard deviation. Asterisks denote statistical difference compared to control (p<0.05 for all conditions). <i>n</i> = 20 for each experimental condition.</p

Representative western blots of p- Akt, total Akt, and β-Actin.

<p>OCs were exposed for 1 hour to either control media (Control) or media containing 5 µM octreotide. P-Akt levels were normalized against total Akt. Octreotide treated levels are expressed as % of control values. P-Akt levels were significantly increased by octreotide treatment (p<0.05). Bars show the mean ± one standard deviation of 3 independent experiments. In each experiment 10 OCs from 5 animals were used.</p

Quantitative analysis of surviving HCs under different experimental conditions in the basal and middle cochlear turns.

<p>No toxic effects were observed in OCs incubated with the AKT inhibitor SH-6 at 10 µM. HC survival was significantly higher in groups treated with octreotide and gentamicin compared to gentamicin treatment alone or treatment with SH-6, octreotide and gentamicin. Asterisks indicate significant difference from treatment with gentamicin only (p<0.01 for all conditions). Data are expressed as the mean number of surviving HCs per 20 inner HCs. Vertical lines represent one standard deviation. <i>n</i> = 6 for each experimental condition.</p

Quantitative analysis of surviving HCs.

<p>(a) Gentamicin-induced HC damage in the OCs of wild-type, SST receptor-1 knockout and SST receptor-1/SST receptor-2 double knockout mice. Photograph of phalloidin-labeled OC. The three outer HC (OHC) rows and a single inner HC (IHC) row can be seen in controls. OCs exposed to 0.5 mM gentamicin demonstrate HC loss. (b) Histograms show a significant difference in the number of surviving HCs in the OCs of SST receptor-1 knockout mice exposed to gentamicin compared to in the gentamicin-treated OCs of wild-type mice (<i>p</i>>0.0001). Histogram and bars represent mean ± one standard deviation. <i>n</i> = 15 for each experimental condition.</p

Effects of peroxisome proliferator activated receptors (PPAR)-γ and -α agonists on cochlear protection from oxidative stress

<div><p>Various insults cause ototoxicity in mammals by increasing oxidative stress leading to apoptosis of auditory hair cells (HCs). The thiazolidinediones (TZDs; e.g., pioglitazone) and fibrate (e.g., fenofibrate) drugs are used for the treatment of diabetes and dyslipidemia. These agents target the peroxisome proliferator-activated receptors, PPARγ and PPARα, which are transcription factors that influence glucose and lipid metabolism, inflammation, and organ protection. In this study, we explored the effects of pioglitazone and other PPAR agonists to prevent gentamicin-induced oxidative stress and apoptosis in mouse organ of Corti (OC) explants. Western blots showed high levels of PPARγ and PPARα proteins in mouse OC lysates. Immunofluorescence assays indicated that PPARγ and PPARα proteins are present in auditory HCs and other cell types in the mouse cochlea. Gentamicin treatment induced production of reactive oxygen species (ROS), lipid peroxidation, caspase activation, PARP-1 cleavage, and HC apoptosis in cultured OCs. Pioglitazone mediated its anti-apoptotic effects by opposing the increase in ROS induced by gentamicin, which inhibited the subsequent formation of 4-hydroxy-2-nonenal (4-HNE) and activation of pro-apoptotic mediators. Pioglitazone mediated its effects by upregulating genes that control ROS production and detoxification pathways leading to restoration of the reduced:oxidized glutathione ratio. Structurally diverse PPAR agonists were protective of HCs. Pioglitazone (PPARγ-specific), tesaglitazar (PPARγ/α-specific), and fenofibric acid (PPARα-specific) all provided >90% protection from gentamicin toxicity by regulation of overlapping subsets of genes controlling ROS detoxification. This study revealed that PPARs play important roles in the cochlea, and that PPAR-targeting drugs possess therapeutic potential as treatment for hearing loss.</p></div