Hearing loss can be caused by a variety of insults, including acoustic trauma and exposure to ototoxins, that principally effect the viability of sensory hair cells via the MAP kinase (MAPK) cell death signaling pathway that incorporates c-Jun N-terminal kinase (JNK). We evaluated the otoprotective efficacy of D-JNKI-1, a cell permeable peptide that blocks the MAPK-JNK signal pathway. The experimental studies included organ cultures of neonatal mouse cochlea exposed to an ototoxic drug and cochleae of adult guinea pigs that were exposed to either an ototoxic drug or acoustic trauma. Results obtained from the organ of Corti explants demonstrated that the MAPK-JNK signal pathway is associated with injury and that blocking of this signal pathway prevented apoptosis in areas of aminoglycoside damage. Treatment of the neomycin-exposed organ of Corti explants with D-JNKI-1 completely prevented hair cell death initiated by this ototoxin. Results from in vivo studies showed that direct application of D-JNKI-1 into the scala tympani of the guinea pig cochlea prevented nearly all hair cell death and permanent hearing loss induced by neomycin ototoxicity. Local delivery of D-JNKI-1 also prevented acoustic trauma-induced permanent hearing loss in a dose-dependent manner. These results indicate that the MAPK-JNK signal pathway is involved in both ototoxicity and acoustic trauma-induced hair cell loss and permanent hearing loss. Blocking this signal pathway with D-JNKI-1 is of potential therapeutic value for long-term protection of both the morphological integrity and physiological function of the organ of Corti during times of oxidative stress.

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Apoptosis is an important process, both for normal development of the inner ear and for removal of oxidative-stress damaged sensory cells from the cochlea. Oxidative-stressors of auditory sensory cells include: loss of trophic factor support, ischemia-reperfusion, and ototoxins. Loss of trophic factor support and cisplatin ototoxicity, both initiate the intracellular production of reactive oxygen species and free radicals. The interaction of reactive oxygen species and free radicals with membrane phospholipids of auditory sensory cells creates aldehydic lipid peroxidation products. One of these aldehydes, 4-hydroxynonenal, functions as a mediator of apoptosis for both auditory neurons and hair cells. We present several approaches for the prevention of auditory sensory loss from reactive oxygen species-induced apoptosis: 1) preventing the formation of reactive oxygen species; (2) neutralizing the toxic products of membrane lipid peroxidation; and 3) blocking the damaged sensory cells' apoptotic pathway.

Hair cell (HC) and supporting cell (SC) productions are completed during early embryonic development of the mammalian cochlea. This study shows that acutely dissociated cells from the newborn rat organ of Corti, developed into so-called otospheres consisting of 98% nestin (+) cells when plated on a non-adherent substratum in the presence of either epidermal growth factor (EGF) or fibroblast growth factor (FGF2). Within cultured otospheres, nestin (+) cells were shown to express EGF receptor (EGFR) and FGFR2 and rapidly give rise to newly formed myosin VIIA (+) HCs and p27(KIP1) (+) SCs. Myosin VIIA (+) HCs had incorporated bromodeoxyuridine (BrdU) demonstrating that they were generated by a mitotic process. Ultrastructural studies confirmed that HCs had differentiated within the otosphere, as defined by the presence of both cuticular plates and stereocilia. This work raises the hypothesis that nestin (+) cells might be a source of newly generated HCs and SCs in the injured postnatal organ of Corti.

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We tested and characterized the therapeutic value of round window membrane-delivered (RWM) D-JNKI-1 peptide (Bonny et al., 2001) against sound trauma-induced hearing loss. Morphological characteristics of sound-damaged hair cell nuclei labeled by Hoechst staining show that apoptosis is the predominant mode of cell death after sound trauma. Analysis of the events occurring after sound trauma demonstrates that c-Jun N-terminal kinase (JNK)/stress-activated protein kinase activates a mitochondrial cell death pathway (i.e., activation of Bax, release of cytochrome c, activation of procaspases, and cleavage of fodrin). Fluorescein isothiocyanate (FITC)-conjugated D-JNKI-1 peptide applied onto an intact cochlear RWM diffuses through this membrane and penetrates cochlear tissues with the exception of the stria vascularis. A time sequence of fluorescence measurements demonstrates that FITC-labeled D-JNKI-1 remains in cochlear tissues for as long as 3 weeks. In addition to blocking JNK-mediated activation of a mitochondrial cell death pathway, RWM-delivered D-JNKI-1 prevents hair cell death and development of a permanent shift in hearing threshold that is caused by sound trauma in a dose-dependent manner (EC 50 ϭ 2.05 M). The therapeutic window for protection of the cochlea from sound trauma with RWM delivery of D-JNKI-1 extended out to 12 h after sound exposure. These results show that the mitogen-activated protein kinase/JNK signaling pathway plays a crucial role in sound trauma-initiated hair cell death. Blocking this signaling pathway with RWM delivery of D-JNKI-1 may have significant therapeutic value as a therapeutic intervention to protect the human cochlea from the effects of sound trauma.