The Hearing Loss In The D-galactose Overdose Model And The Relating Mechanism

Part I Comparison of three methods forisolation of nucleic acids from rat's membranate inner ear tissueObjective To compare three methods for extraction of nucleic acid from rat'smembranate inner ear tissue.Material and methods Method of alkaline denaturation, a conventionalphenol-chloroform method and method with Trizol reagent were respectively used to extract the slight nucleic acid from rat's membranate inner ear tissue. We assess the amount and quality of nucleic acid using a UV-spectrometer and polymerase chain reaction (PCR).Results The yield and purity (OD260/OD280) of DNA from inner ear tissueusing the phenol-chloroform method was the highest of the three. Mitochondrial DNA(MtDNA) fragment can be PCR amplified from nucleic acid prepared by all methods, while no nuclear DNA(nDNA) fragment can be amplified from nucleic acid prepared by method of alkaline denaturation. Both nuclear and mitochondrial genes could be amplified by RT-PCR from the RNA prepared by Trizol reagent. Conclusion Adequate amount and high-quality of mtDNA, nDNA and RNA areobtained from unilateral rat's membranate inner ear tissue. Method of alkaline denaturation could be chosen when we need mtDNA without nDNA, while phenol-chloroform method was suitable for extracting total DNA(including nuclear DNA and mtDNA); method with Trizol reagent was suitable for extracting total RNA and total DNA.Part II The D-galactose induced aging modelingand the relating mtDNA deletionsObjective To investigate the changes in inner ear enzymatic activity, theoccurring of mtDNA 4,834bp deletion in inner ear and the relating hearing loss after two dosage of D-galactose (D-gal) injection into rats. Furthermore, the incidence of the mitochondrial DNA (mtDNA) 4,834bp deletion and other deletions in different tissues was compared.Material and methods The animal models were induced by dailysubcutaneous injection of D-gal (150mg/kg/d, 50mg/kg/d) into rats. Activities of GSH-P_X and Na-K ATPase of rats' inner ear tissues were measured using test kits, mtDNA deletions were examined by nested-PCR, and the hearing loss was judged byABRtest.Results The activities of GSH-Px and ATPase decreased significantly in the rats'inner ear tissues in the two D-gal treated groups, especially in the high dose D-gal group, as compared to those in the control group. At the mean time, remarkable increases were observed in the mtDNA 4,834bp deletion and 7,506bp deletion rate in inner ear tissues in the two D-gal treated groups, just as it was in other tissues, especially in high dose D-gal group, as compared to those in the control group. However, the relating hearing loss was nearly identical in the 3 groups. Blood samples were used to show the lowest incidence of 4,834bp deletion and 7,506bp deletion among the selected tissues.Conclusion Daily subcutaneous injections of D-gal into rats for 8 weeks couldlead to biochemical defects and mtDNA 4,834bp deletion and mtDNA 7,506bp deletion of inner ear tissue, but the mtDNA deletions alone is probably not sufficient to produce the deamess phenotype in our model. Using blood samples might lead to a "false negative" in the PCR examination of mtDNA 4,834bp and 7,506bp deletion in clinical research. A higher sensitivity could be found using biopsy and nested-PCR to examine mtDNA 4,834bp and 7,506bp deletion.Part III The relation between the D-galactose induced large fragment deletion of mtDNA in inner ear and the risk to aminoglycoside antibiotic induced deafnessObjective To explore the relation between the D-galactose (D-gal) induced largefragment deletion of mtDNA in inner ear and the risk to aminoglycoside antibiotic(AmAn) induced deamess.Materials and Methods The animal models were induced by application oftwo dose of D-gal (150mg/kg/d, 50mg/kg/d) before kanamycin sulfate into rats . Activities of GSH-P_X and Na-K ATPase of rats' inner ear tissues were measured using test kits, mtDNA deletions were examined by nested-PCR, and the hearing loss was judged by ABR test.Result It was found that the activities of GSH-P_X and Na-K ATPase decreased in inner ear of the rat treated with D-galatose than of the control. However, there was no significant difference in elevation of ABR threshold between the rat with mtDNA4834 deletion induced by D-galatose and control. However, after aminoglycoside antibiotic injected, the hearing threshold of the rats carrying mtDNA 4,834bp deletion and 7,506bp deletion increased significantly compare to that of the rats without this deletion.Conclusions The rat could be induced resembled accelerated aging in inner earby injecting D-galactose. Moreover, it suggests that mtDNA4834 deletion don't induce the hearing loss directly, but can greatly enhance the sensitivity of the inner ear to the aminoglycoside antibiotic as a predisposing factor.Part I V The role of nuclear factors in the aminoglycoside hypersensitivity induced deafnessObjective To study the changes of mtDNA(mitochondrial DNA) CN(copynumbers) and mRNA levels of MTFA(mitochondrial transcription factor A) and ANTl(adenine nucleotide translocases 1) in the inner ear tissues of rats with high sensitivity to aminoglycoside antibiotic ototoxicity.To investigate the possible role of these nuclear factors in the process.Materials and methods The animal models were induced by intraperitonealinjection of D-galactose (50mg/kg/d,150mg/kg/d) to become more sensitive to aminoglycoside antibiotic(AmAn),mtDNA copy numbers were detected by the multipler PCR, mRNA levels of MTFA, ANT1, COXIIIand NDII were measured by semi-quantitive reverse transcription polymerase chain reaction(RT-PCR), the sensitivity to AmAn was judged by the threshold-shift(TS) of ABR. And the large fragment deletion mutation of mtDNA was examined by nested-PCR.Result The mtDNA CN: A group:4.34±1.26, B 组 7.04±1.54. The mRNAlevels of MTFA: 1.87 ± 0.33 of A group versus 4.34±1.79 of B group; mRNA levels of ANT1: 6.05 ± 1.62 of A group versus 3.35 ± 0.85 of B group; mRNA levels of COXIII: 2.43 ± 0.74 of A group versus 2.61±0.83 of B group; mRNA levels of NDII: 2.64 ± 0.53 of A group versus 2.77 ± 0.61 of B group. Both the mtDNA CN and the mRNA levels of MTFA of A group were significantly lower than that of B group. But the mRNA levels of ANT1 were much higher than that of B group (P<0.05) . And the levels of COXIII and ND II mRNA levels were nearly identical in the two groups. The TS after AmAn injection.were 73.54±11.28 dBpeSPL in A group and 40.63 ± 9.04 dBpeSPL in B group, The TS in A group was higher than B group(p<0.05). Furthmore, the mtDNA 4834bp deletion rate of inner ear tissues in group A was remarkably higher than the mtDNA 4834bp deletion rate in group B(62.5% versusConclusion There were possible roles of several nuclear factors of inner ear tissue in the aminoglycoside hypersensitivity induced deafness.