Nogo in the Mammalian cochlea.

HYPOTHESIS: Different members of the Nogo system are expressed in the mammalian cochlea. BACKGROUND: The protein Nogo has gained a lot of attention during the last couple of years because it inhibits neurite outgrowth in the adult central nervous system. In contrast to the central nervous system, very little is known regarding the expression and possible function of the Nogo system within the inner ear. METHODS: Using reverse-transcriptase-polymerase chain reaction and immunohistochemistry, we analyzed for the expression of members of the Nogo system within the cochlea. In addition, we determined hearing levels of Nogo A knockout and wild-type mice with auditory brainstem response audiometry. RESULTS: In this study, we demonstrate the expression of Nogo A, B, C, and of Nogo receptor mRNA in the organ of Corti, spiral ganglion, and stria vascularis. Immunohistochemistry revealed that Nogo A and Nogo receptor localize to the spiral ganglion neurons. Interestingly, Nogo A expression was also observed in the outer and inner hair cells of the organ of Corti. As revealed by light microscopy, deletion of Nogo A does not alter cochlear microanatomy. We have assessed hearing levels in 10-month old wild-type and Nogo A knockout mice, and thereby, we could not detect any differences between these 2 groups. CONCLUSION: Different members of the Nogo family are expressed in the mammalian cochlea. Deletion of Nogo A does not alter cochlea microanatomy or hearing levels compared with wild-type mice.

Somatostatin and gentamicin-induced auditory hair cell loss.

OBJECTIVE/HYPOTHESIS: Hair cells of the mammalian auditory system do not regenerate, and therefore their loss leads to irreversible hearing loss. Aminoglycosides, among other substances, can irreversibly damage hair cells. Somatostatin, a peptide with hormone/neurotransmitter properties, has neuroprotective effects by binding to its receptor. In this study, we tested whether somatostatin can protect hair cells from gentamicin-induced damage in vitro. STUDY DESIGN: This study confirmed the expression of somatostatin receptor mRNA within the cochlea and analyzed the effect of somatostatin on gentamicin-induced hair cell damage and death in vitro. METHODS: Expression of somatostatin receptor mRNA in the rat cochlea was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Protection of auditory hair cells from gentamicin was tested using two different concentrations (1 microM and 5 microM, respectively) of somatostatin. RESULTS: We detected somatostatin receptor-1 and -2 mRNA and in the organ of Corti (OC), spiral ganglion, and stria vascularis by RT-PCR. Moreover, we could see significantly less hair cell loss in the OCs that were pretreated with either 1 microM or 5 microM of somatostatin as compared with samples treated with gentamicin alone. CONCLUSIONS: Decreased hair cell loss in somatostatin-treated samples that had been exposed to gentamicin provides evidence for a protective effect of somatostatin in aminoglycoside-induced hair cell death in vitro.

Resveratrol protects auditory hair cells from gentamicin toxicity.

Resveratrol is a naturally occurring polyphenol that is synthesized by a variety of plant species. It is abundant in grapes and grape products (e.g., red wine). Resveratrol has demonstrated reactive oxygen species (ROS) scavenger activity, and it has been linked to nuclear factor-kappa B (NF-kappaB) activity. We recently demonstrated that NF-kappaB is important to the survival of immature mammalian hair cells. Therefore, we undertook an in vitro experiment to determine if resveratrol is able to exert some protective influence against gentamicin-induced damage to and death of auditory hair cells. To accomplish this, we dissected the organ of Corti (OC) from newborn Sprague-Dawley rats and cultured the OCs in medium overnight for recovery. We treated two groups of OC explants with different concentrations of resveratrol plus gentamicin for 24 hours; for comparison and control purposes, we also treated a group of explants with gentamicin only and we left a group untreated. We found that resveratrol in both concentrations had a moderate but statistically significant protective effect against gentamicin-induced toxicity in vitro.

NF-kappaB-dependent apoptotic hair cell death in the auditory system.

Hair cells are the most vulnerable elements in the inner ear and their degeneration is the most common cause of hearing loss. In the last few years progress has been made in uncovering the molecular mechanisms involved in hair cell damage and death. However, little is known about factors important for hair cell survival. Recently, it has been demonstrated that the transcription factor NF-kappaB is required for survival of immature auditory hair cells in vitro. Here we used DNA microarray technology to explore NF-kappaB downstream events in organ of Corti explants of postnatal day-5 Sprague-Dawley rats which were exposed to a cell-permeable NF-kappaB-inhibitory peptide. Gene expression was analyzed using DNA microarray technology. Genes were selected on the basis of comparative analysis, which reliably distinguished the NF-kappaB inhibitor-treated samples from control samples. Interestingly, among the up-regulated genes was the gene coding for the regulatory subunit of phosphatidylinositol 3-kinase. Moreover, inhibition of the phosphatidylinositol 3-kinase signaling pathway in organ of Corti explants exposed to the NF-kappaB inhibitor reduced caspase-3 activation. These data link NF-kappaB-dependent hair cell death to phosphatidylinositol 3-kinase signaling.

The effect of erythropoietin on gentamicin-induced auditory hair cell loss.

OBJECTIVE/HYPOTHESIS: Mammalian auditory hair cells that are unable to regenerate and various agents, including gentamicin, can irreversibly damage the hair cells. Erythropoietin, known as the primary regulator of erythropoiesis, exerts also neuroprotective effects by binding to its receptor. We tested whether erythropoietin can protect the hair cells from gentamicin-induced damage. STUDY DESIGN: This study localized the erythropoietin receptor in the cochlea and analyzed the effect of erythropoietin on gentamicin-damaged hair cells in vitro. METHODS: Expression of erythropoietin receptor in the rat cochlea was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Protection of auditory hair cells from gentamicin was tested in vitro by exposing cultured rat organs of Corti with increasing concentrations of erythropoietin (0.1 U/mL, 1 U/mL, and 10 U/mL). RESULTS: We detected erythropoietin and erythropoietin receptor mRNA expression in the organ of Corti, spiral ganglion, and stria vascularis by RT-PCR. Immunohistochemistry revealed that the erythropoietin receptor localizes to the outer and inner hair cells and supporting cells of the organ of Corti, as well as to the spiral ganglion cells and the stria vascularis. Significantly less hair cell loss occurred in the organs of Corti that were pretreated with 0.1 U/mL erythropoietin as compared with samples treated with gentamicin only. CONCLUSION: Decreased hair cell loss in erythropoietin-treated organs of Corti that had been exposed to gentamicin provides evidence for a protective effect of erythropoietin in aminoglycoside-induced hair cell death.

NF-kappaB is required for survival of immature auditory hair cells in vitro.

Damage to auditory hair cells in the inner ear as a consequence of aging, disease, acoustic trauma, or exposure to ototoxins underlies most cases of hearing impairment. Because the mammalian ear cannot replace damaged hair cells, loss of hearing is irreversible and progressive throughout life. One of the current goals of inner ear biology is to develop therapeutic strategies to prevent hair cell degeneration. Although important progress has been made in discovering factors that mediate hair cell death, very little is known about the molecular pathway(s) that signal survival. Here we considered the role of NF-kappaB, a ubiquitous transcription factor that plays a major role in the regulation of many apoptosis- and stress-related genes, in mediating hair cell survival. NF-kappaB was detected in a constitutively active form in the organ of Corti of 5-day-old rats. Selective inhibition of NF-kappaB through use of a cell-permeable inhibitory peptide in vitro caused massive degeneration of hair cells within 24 h of inhibitor application. Hair cell death occurred through an apoptotic pathway through activation of caspase-3 and may involve transcriptional down-regulation of the gadd45beta gene, an anti-apoptotic NF-kappaB target. In view of our results, it seems likely that NF-kappaB may participate in normal hair cell function.