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

Objective To investigate the ototoxic effects of streptomycin in vestibular organotypic cultures. Methods F344 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 ceils was stained with fluorescent phalloidin, and its nucleus was stained with topro-3 DNA probe. The vestibular hair cells were quantitatively counted and photographed under confocal fluorescent microscope. Results Morphological 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. wenty-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. Conclusion Streptomycin 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.

Objective To explore the quantitative relationship between the reduction of distortion product otoacoustic emission (DPOAE) and the percentage of outer hair cell loss. Methods Coadministration of cisplatin (0.2 mg/kg) and ethacrynic acid (40 mg/kg) were used to estabhsh 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. Results Cisplatin 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). Condusions It may be helpful to evaluate missing percentage of outer hair cells from reduction in DPOAE levels.

Objective To evaluate if caspase pathway was involved in streptomycin-induced cell apoptosis in cochlear hair cells. Methods F344 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. Results Streptomycin with 1 nunol/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. Conclusions Apoptosis 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.

Objective:To measure the cochlear compound action potential (CAP) and the densities of hair cells (HCs) along the whole length of the basilar membrane (BM) in adult chinchillas. And to investigate the relationship between the severity of inner hair cells (IHCs) loss and the changes of CAP by using carboplatin-cochlear lesion model.Methods:Totally 18 chinchillas were recruited after ontological evaluation. They were randomly divided into three groups (with 6 subjects in each), A: control, B and C: legion groups treated with one or two shot(s) of carboplatin respectively (76 mg/kg in one shot, i.p., one-week interval between the two shots). Endpoint tests were performed 30 days after the carboplatin treatment in groups B and C, and matched time in group A. A sliver-ball electrode was placed into round window niche via hypotympanic approach in anesthetized chinchilla. CAP was measured in response to clicks and tone burst of 0.5, 1, 2, 4, 8, 16 kHz respectively under anesthesia. CAP amplitudes and thresholds were measured and compared across the groups. After the recording, the whole cochlea surface preparation was made and the HCs were stained in histochemistry against substrate of succinate dehydrogenase (SDH). Images were taken with high-resolution digital camera under light microscope and across the whole cochlea. The length of the basilar membrane (BM) and the number of both IHCs and OHCs were counted. The HC density was calculated as the number of HCs per 10% BM length.Results:The CAP thresholds were (7.1±2.6), (25.4±5.0), (24.6±5.4), (10.4±5.0), (0.4±1.4), (4.2±6.3) and (17.1±14.1) dB SPL (from 6 subjects in group A, n=12 ears) corresponding to stimuli of Click and 0.5, 1, 2, 4, 8, 16 kHz tone bursts respectively. The total number of cochlear HCs were measured as (8 936±643) ( ± s) and the average length of the BMs was (17.73±1.012) mm from the six subjects in the group A ( n=12 ears). The HC density was found to be varied slightly across the BM. There was no significant CAP threshold difference between the control (group A) and the group B, which received one shot of carboplatin. However, the maximal CAP amplitude was reduced by 40% in the group B and compared with group A. Correspondingly, approximately 40% loss of IHCs were seen. In contrast, a significant CAP threshold shift was seen in subjects receiving two shots of carboplatin (group C), which was accompanied by a loss of 90% IHCs. Conclusions:The CAP thresholds of adult chinchillas show typical open-V shape with the lowest values at 2, 4, and 8 kHz. IHC loss by carboplatin in certain degree is well correlated with CAP amplitude reduction, but does not change the threshold when inner hair cell loss reaches 40%, however, if inner hair cell loss exceeds 80%, the threshold shift of CAP will be inevitable.