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Oxaliplatin, an anticancer drug commonly used to treat colorectal cancer and other tumors, has a number of serious side effects, most notably neuropathy and ototoxicity. To gain insights into its ototoxic profile, oxaliplatin was applied to rat cochlear organ cultures. Consistent with it neurotoxic propensity, oxaliplatin selectively damaged nerve fibers at a very low dose 1 μM. In contrast, the dose required to damage hair cells and spiral ganglion neurons was 50 fold higher (50 μM). Oxailiplatin-induced cochlear lesions initially increased with dose, but unexpectedly decreased at very high doses. This non-linear dose response could be related to depressed oxaliplatin uptake via active transport mechanisms. Previous studies have demonstrated that axonal degeneration involves biologically active processes which can be greatly attenuated by nicotinamide adenine dinucleotide (NAD+). To determine if NAD+ would protect spiral ganglion axons and the hair cells from oxaliplatin damage, cochlear cultures were treated with oxaliplatin alone at doses of 10 μM or 50 μM respectively as controls or combined with 20mM NAD+. Treatment with 10 μM oxaliplatin for 48hours resulted in minor damage to auditory nerve fibers, but spared cochlear hair cells. However, when cochlear cultures were treated with 10 μM oxaliplatin plus 20mM NAD+, most auditory nerve fibers were intact. 50 μM oxaliplatin destroyed most of spiral ganglion neurons and cochlear hair cells with apoptotic characteristics of cell fragmentations. However, 50 μM oxaliplatin plus 20mM NAD+ treatment greatly reduced neuronal degenerations and hair cell missing. The results suggested that NAD+ provides significant protection against oxaliplatin-induced neurotoxicity and ototoxicity, which may be due to its actions of antioxidant, antiapoptosis, and energy supply.

Lead is a major environmental toxicant throughout the world. Lead can induce severe neurotoxicity including irreversible hearing impairment. Many in vivo studies have shown that lead damages the auditory nervous system, but has little or no effect on cochlear sensory hair cells. To gain insights on lead ototoxic and neurotoxic effects in vitro, lead acetate (LA) was applied to postnatal day 3–4 rat cochlear organotypic cultures for 24 or 72 h with doses of 0.1, 0.5,1, 2 or 4 mM. After 24 or 72 h treatment with lead acetate, nearly all of cochlear sensory hair cells were intact. However, after 72 h treatment, the peripheral auditory nerve fibers projecting to the hair cells and the spiral ganglion neurons (SGN) were damaged when lead concentration exceeded 2 mM. Our results indicated that 72 h treatment with only the high doses (> 2 mM) of lead actate damaged SGNs and peripheral nerve fibers; hair cells remained structurally intact even after 4 mM treatment. These results show that lead primarily damages cochlear nerve fibers andSGNratherthanhaircells.

Cisplatin belongs to platinum-based drugs and is widely used in cancer chemotherapy. Ototoxicity is one of the major dose limiting side-effects of cisplatin. For toxicity to occur cisplatin must first be transported from the bloodstream into cochlear cells. Three copper transporters are considered pathways for regulating the uptake and translocation of cisplatin into cells: Ctr1, ATP7A and ATP7B. Our recent study with cochlear organotypic cultures shows that cochlear hair cells can be destroyed by cisplatin at low concentrations from 10μm to 100μn. However, high doses of cisplatin cannot damage hair cells, maybe due to intrinsic feedback reactions that increase export of platinum by ATP7B when the platinum concentration is high in extracellular space. Cimitidine is a specific copper transporter inhibitor that can block the entrance of copper and platinum, and may prevent cisplatin-induced cochlear hair cell injury. To evaluate this hypothesis, we treated cochlear organotypic cultures with cisplatin (10 μm or 50 μm) alone, or cisplatin combined with cimitidine at concentrations ranging from 10-2000 μm for 48 hours. cisplatin at 10 μm damaged about 20% hair cells. In contrast, when cimitidine (10 μm, 100 μm and 2000 μm) was added to the culture, near 100% cochlear hair cell survived. At higher concentration (50 μm), cisplatin destroyed about 80% of cochlear hair cells. However, 100 μmcimitidine rescued about 50% hair cells from cisplatin damage, and 2000μm cimitidine protected about 80% hair cells. The data of western blot showed that CTR1 and ATP7B expressions were increased in cisplatin treated cochlear tissue, but cimitidine significantly reduced CTR1 and ATP7B. In addition, ATP7A expression was depressed a little after cisplatin treatment. Considering that Ctr1 is involved in copper and platinum influx, but the ATP7A and ATP7B are copper export transporters, the results suggest that cimitidine can effectively block the entrance by copper transporters and stop the influx of cisplatin.

Objective To investigate the occurrence and possible mechanisms of apoptosis in cochlear epithelium and spiral ganglion neurons after mefloquine treatment. Methods We used quantitative RT–PCR apoptosis–focused gene arrays (96–well, 84 apoptosis related genes) to assess changes of gene expression in the cochlear basilar membrane (hair cells–supporting cells) and spiral ganglion neurons of rat cochlear organotypic cultures treated with 100 μM mefloquine for 3 h. Results Significant up–or down–regulation in gene expression was detected in 23 genes in the cochlear basilar membrane, and in 32 genes in the spiral ganglion neurons compared with time–matched controls. The responding genes could be classified as pro–or anti–apoptotic, and were mainly implicated in the Bcl–2, Caspase, Card, IAP, TNF ligand/TNF receptor, Death domain/Death effector domain, DNA damage/p53, and NF–kappa B families. Synthetic analysis suggested that these families could be revised to two major pathways mainly involved in the death receptor–mediated signaling pathway and apoptotic mitochondrial pathway. In addition, it was found that numerous anti–apoptotic genes such as Bcl2a1, Birc1b, Birc3, Birc4, Bnip1, Cflar, Il10, Lhx4, Mcl1, Nfkb1, Prlr, Prok2, and TNF were greatly up–regulated in the cochlear tissue, which might imply the co–existence of protective response in the cells at the early stage of mefloquine–induced damage.

Abxtract Cisplatin damages cochlear hair cells and spiral ganglion neurons through cell death signaling pathways that are not fully understood. We used focused apoptosis gene microarrays to study early changes in gene expression in cochlear cultures from P3 neonatal rats treated with cisplatin (0.2 mM). After 12 hours of cisplatin treatment, more than 50% of the 96 genes on the array showed a significant decrease in expression, consistent with widespread cell death. However, after 3 hours of cisplatin treatment, 10 genes showed significant increase in expression in total cochlear tissue. In experiments with subsets of cochlear tissues, at 3h, cisplatin induced increased expression of 12 genes in the cochlear sensory epithelium (basilar membrane) and 11 genes in the spiral ganglion (tissue of Rosenthal’s canal, containing the spiral ganglion). These included pro– and anti–apoptotic genes involved in the p53 signaling pathway, TNF receptor family, NF–kappaB pathway, death domain family, death effector domain family, Bcl–2 family, CARD family, TRAF family, and GTP signal transduction. Although the changes in gene expression showed an overlap between basilar membrane and spiral ganglion, other changes, which may reflect the unique response of each tissue, were also observed. Pifithrin–a blocked cisplatin–induced up–regulation of genes in the p53 signaling pathway when assayed by both superarray and real time PCR. The data add to our understanding of the involvement of p53 in cisplatin–induced ototoxicity and otoprotection, conferred by the p53 inhibitor Pifithrin–a.

Cells in mammalian cochleae virtually stop proliferation and exit cellular circle before birth. Consequently, hair cells and spiral ganglion neurons destroyed by ototoxic factors cannot be replaced through proliferative regeneration. However, substantial proliferation occurs in organotypic cultures of cochleae from postnatal mice. In the present study, we studied the time course of proliferative growth in cultures of mouse cochlea explants obtained from up to 12 postnatal days. The mitotic nature of this growth was confirmed by bromodeoxyuridine (BrdU) staining and expression of proliferation cell nuclear antigen (PCNA) evaluated with real–time quantitative polymerase chain reaction (RT–PCR). Similar growth time course was found in the cochlear explants of different postnatal ages. The new growth reached its maximum at around 2 days in culture followed by a slow–down, and virtually stopped after 5 days of culture. The possible mechanisms and the significance of this proliferation are discussed.