OBJECTIVE Exploration of project is to introduce a cochlear organotypic culture technique and the histological evaluation of cochlear hair cells, auditory nerve fibers, and spiral ganglion neurons from rat on post-natal day 3. METHODS The cochlear basilar membrane was placed on the surface of rattail collagen gel in culture medium. The cochlear specimens were double-labeled with FITC-conjugated phalloidin for showing the stereocilia and cuticular plate of the hair cells plus a monoclonal antibody against neurofilament 200 for showing the spiral ganglion neurons and their auditory nerve fibers. Specimens were observed under a Confocal Laser Scanning Microscope with appropriate filters. RESULTS Following the culturing period of 1 to 3 days, cochlear inner and outer hair cells presented normal without any contabescence or missing. The auditory nerve fibers were in an orderly arrangement and the morphology of spiral ganglion neurons were also in good shape. CONCLUSION The cochlear organotypic culture technique and the histological examinations will be helpful to evaluate the cochlear hair cells and the spiral ganglion neurons in various experimental models in vitro.

OBJECTIVESTo study the status of cochlear mitochondrial DNA (mtDNA) and to determine the location of mtDNA deletion in aged mice.

METHODSWe detected cochlear mtDNA in 2, 7 - 10 and 17 - 19 month old mice by nested polymerase chain reaction (PCR) and DNA sequencing.

RESULTSmtDNA3867bp deletions were found in the cochleae of aged mice. The deletion occurred within nt9103-nt12970 and were flanked by 15 base pair direct repeats. Comparing the incidence of mtDNA3867bp deletions, 17 - 19 month old mice (7/8) were significantly higher than 7 - 10 month old mice (4/16). The deletion was not observed in 2 month old mice (0/7). The ratio of deleted mtDNA/total mtDNA in 17 - 19 month old mice was higher than in 7 - 10 month old mice (P < 0.001).

CONCLUSIONCochlear mtDNA 3867bp deletion in aged mice may be related to presbycusis.

Aging ; genetics ; Animals ; Base Sequence ; Cochlea ; metabolism ; DNA, Mitochondrial ; analysis ; genetics ; Mice ; Molecular Sequence Data ; Oxidative Phosphorylation ; Presbycusis ; etiology ; Sequence Deletion

OBJECTIVESTo investigate the tissue specificity of reactive oxygen species (ROS) damage to mitochondrial DNA (mtDNA) and to determine whether cochlear mtDNA is a sensitive target for ROS damage.

METHODS10 Cu/ZnSOD gene (Cu/Zn superoxide dismutase gene, Sod1) knockout mice and 16 wild-type mice were analyzed by nested polymerase chain reaction (PCR).

RESULTSThree deletions were detected in various tissues of Sod1 knockout mice. MtDNA3867bp and mtDNA3726bp deletions were the most visible, and mtDNA4236bp deletion was barely detected in these tissues. There were obvious differences in the ratio of deleted mtDNA/total mtDNA in different tissue. Deleted mtDNA was most abundant in the liver and kidney and less in cochlea, heart and brain. The lowest was in spleen and skin. The ratio in various tissues was 3 - 20 times in Sod1 knockout mice over wild-type mice. In cochlea, the ratio was about 15.

CONCLUSIONSWithout the protection of Sod1, ROS can lead to mtDNA deletions in various tissues with significant tissue specificity. Cochlear mtDNA is a sensitive target for ROS damage.

Animals ; Base Sequence ; Brain ; metabolism ; Cochlea ; metabolism ; DNA Mutational Analysis ; DNA, Mitochondrial ; chemistry ; genetics ; Kidney ; metabolism ; Liver ; metabolism ; Mice ; Mice, Inbred Strains ; Mice, Knockout ; Molecular Sequence Data ; Myocardium ; metabolism ; RNA, Ribosomal ; genetics ; Sequence Deletion ; Skin ; metabolism ; Spleen ; metabolism ; Superoxide Dismutase ; genetics

OBJECTIVETo evaluate if caspase pathway was involved in streptomycin-induced cell apoptosis in cochlear hair cells.

METHODSF344 rats at postnatal day 3 or 4 were used for the study in cochlear organotypic cultures. The cochlear basilar membrane was micro-dissected out and cultured overnight, and then treated with 1 mmol/L streptomycin for 24 hours. Before the termination, the activity of caspase-8, 9 or 6 were detected with FAM-peptide-FMK labeled caspase-8, 9 or 6, respectively. The stereocilia and cuticular plate of hair cells were stained with TRITC conjugated phalloidin, and the nuclei were stained with Topro-3 DNA probe. The specimens were observed and photographed under confocal fluorescent microscope.

RESULTSStreptomycin with 1 mmol/L causes about 80% cochlear hair cells missing in the basal turn and 10% hair cell loss in the apex. After streptomycin treatment, the nuclear shrinkage and fragmentation were found in most cochlear hair cells, and the caspase-8, caspase-9 and caspase-6 were greatly activated.

CONCLUSIONSApoptosis is involved in the cochlear hair cells death induced by Streptomycin in vitro. The caspase activities in upstream and downstream are maybe the major apoptotic pathway.

Animals ; Apoptosis ; drug effects ; Caspase 6 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Cells, Cultured ; Hair Cells, Auditory ; cytology ; drug effects ; Rats ; Rats, Inbred F344 ; Streptomycin ; adverse effects

OBJECTIVETo explore the quantitative relationship between the reduction of distortion product otoacoustic emission (DPOAE) and the percentage of outer hair cell loss.

METHODSCoadministration of cisplatin (0.2 mg/kg) and ethacrynic acid (40 mg/kg) were used to establish a cochlear lesion model in chinchillas. DPOAE was measured before and 1 week, 2 weeks, and 3 weeks later respectively after cisplatin and ethacrynic acid treatment. Animals were terminated 3 weeks after the treatment. Cochlear surface preparations were performed, and the cochlear hair cells were counted through entire length of the cochlea. The correlation between DPOAE reduction and outer hair cell missing was analyzed using Pearson correlation analysis.

RESULTSCisplatin and ethacrynic acid treatment induced cochlear hair cell lesion that the outer hair cell loss in the cochlea developed in a stereotypic pattern; damage began in the base of the cochlea and progressed towards the apex. Reduction of DPOAE was relatively consistent with outer hair cells loss. On the average, 1% outer hair cells loss may result in 0.24 dB reduction in DPOAE levels. Pearson analysis showed a positive correlation between the reduction in DPOAE and missing of outer hair cells (r = 0.796, P < 0.05).

CONCLUSIONSIt may be helpful to evaluate missing percentage of outer hair cells from reduction in DPOAE levels.

Animals ; Cell Count ; Chinchilla ; Cisplatin ; Disease Models, Animal ; Ethacrynic Acid ; Hair Cells, Auditory, Outer ; cytology ; pathology ; Otoacoustic Emissions, Spontaneous

OBJECTIVETo investigate the ototoxic effects of streptomycin in vestibular organotypic cultures.

METHODSF344 rats with age at postnatal day three or four were used for this study. The maculae of saccule and utricle were routinely dissected out and cultured with serum-free medium containing various dose of streptomycin for 24 hours. The ciliary turf of vestibular hair cells was stained with fluorescent phalloidin, and its nucleus was stained with to pro-3 DNA probe. The vestibular hair cells were quantitatively counted and photographed under confocal fluorescent microscope.

RESULTSMorphological feature of vestibular hair cells were good in normal control cultures. However, the density of hair cells was decreased in evidence with increase of streptomycin sulfate concentrations. Twenty-four hours after streptomycin cultures, 0.25 mmol/L streptomycin caused a 10% hair cell missing, 50% hair cell loss from 1 mmol/L streptomycin treatment, and more than 75% hair cells gone post-3 mmol/L streptomycin cultures. After streptomycin treatment, the nuclear shrinkage and fragmentation were found in vestibular hair cells, whereas the vestibular supporting cells were normal.

CONCLUSIONStreptomycin induced-vestibular hair cells lesion was in a dose dependent manner with nuclear shrinkage and fragmentation. This may suggest that streptomycin leads vestibular hair cell die through apoptosis.

Animals ; Apoptosis ; drug effects ; Hair Cells, Vestibular ; cytology ; drug effects ; Organ Culture Techniques ; Rats ; Rats, Inbred F344 ; Streptomycin ; adverse effects

【Objective】To establish a reliable and reproducible mouse model of cisplatin induced inner ear hair cells loss and to make possible for further study of mechanisms of cisplatin-induced ototoxicity in mice from the molecular and genetic aspects. 【Methods】Twenty healthy mice were evenly divided into two groups in random. The mice in group A received a single dose of furosemide（200 mg/kg，intraperitoneal）followed by a single dose of cisplatin（1.0 mg/kg，intraperitoneal）1 h later，and the mice in group B received a single dose of furosemide（200 mg/kg，intraperitoneal）followed by a single dose of cisplatin（0.5 mg/kg，intraperitoneal）1 h later. Auditory brainstem response（ABR）and distortion product otoacoustic emission （DPOAE） were tested in both groups 10 d post the cisplatin-furosemide treatment and bilateral cochlea were taken out and fixed，one side cochlea were used for the surface preparation of total cochlear basal membrane and stained to count the number of cochlear hair cells from apex to base，the other was embedded with EPON 812 gel and then cut into half thin slices. Six mice were used as control group. 【Results】In both groups，the average ABR threshold of mice at each frequency were elevated，while the average DPOAE values were decreased. The average ABR threshold in group A increased significantly and the amplitude of each frequency decreased a lot，and DPOAE in high frequencies vanished；In group B，there was a minor elevation of ABR threshold with a slightly lower amplitude in higher- frequencies（16，20，32 kHz）but no amplitude change in lower frequencies（4，8，12 kHz）both in ABR and DPOAE. The surface preparation of Cochlear results in group A showed that nearly all the outer hair cells in the base had been destroyed，and most were destroyed in the apex；In group B，only the majority of cochlear outer hair cells at the base missed. There was no decrease of the numbers of the inner hair cells in both groups. Semi-thin cross-sections results also showed that the stria vascularis and spiral ganglion cells were close to normal in both groups. 【Conclusion】Co- administration of single dose of 1.0 mg/kg cisplatin with a single dose of 200 mg/kg furosemide may induce significant loss of cochlear outer hair cells in mice.