Protection Of Cochlea From Aminoglycoside Ototoxicity With Manganese Superoxide Dismutase Gene In Aging Rat

Partâ… Study of gene transfer into rat cochlea using different vectors in vivo and in vitroObjective Plasmids,recombinant adenoviruses (rAds), and recombinant adeno- associated viruses (rAAVs) are frequently used vectors,each with distinctive characteristics that may be advantageous in cochlear gene therapy. Although the general advantages and disadvantages of these vectors have been evaluated, the existing information is incomplete and no data are available on the delivery of transgenes to primary cultured marginal cells from the stria vascularis using nonviral and viral vectors. Methods In this study,a plasmid vector,rAd5,and rAAV2 were used to transfect rat cochlear marginal cells of stria vascularis by injection into the perilymph through the round window membrane,and the transfection efficiency, target tissue accessibility, cell/ tissue toxicity, time course of expression, and effect on hearing were evaluated in vivo and in vitro. Results We found that rAd5 had high transfection efficiency and low cytotoxicity in vitro, and rAAV2 produced the long-time stable expression and had the lowest vector-related ototoxicity in vivo. Conclusions rAd5 was more suitable for transgenic research of stria vascularis cells in vitro,while rAAV2 was a better vector for cochlear tissue transfection in vivo. Partâ…¡Construction and expression of rAAV2-IRES-EGFP/ MnSOD in vivo and in vitroObjective Construct recombinant adeno-associated viral vector 2-IRES-EGFP/ MnSOD (rAAV2-IRES-EGFP/MnSOD) and make it express in primary cultured marginal cells of the stria vascularis and cochlea tissue of the rats. Methods The aimed segments were obtained from rat myocardial tissue,which were inserted into a eukaryotic expression plasmid pEGFP-N1/MnSOD or pSNAV2.0-IRES-EGFP/MnSOD, then the production of recombinant adeno-associated viral vector 2-IRES-EGFP/MnSOD (rAAV2-IRES-EGFP/ MnSOD) particles were made.The expression of interested gene was detected by co-Focus Fluorescence microscopy, fluorescence activated call sorting (FACS),PCR and Westernblot. Results Rat MnSOD cDNA of full sequence was detected in eukaryotic expression plasmid pEGFP-N1/MnSOD and pSNAV2.0-IRES-EGFP/MnSOD. MnSOD protein detected in the marginal cells and cochlea tissue after transfection was higher than the control. Conclusion Recombinant virus, rAAV2-IRES-EGFP/MnSOD have been successfully constructed and expressed in marginal cells and cochlea tissue of the rat.This research paved the way for antioxidant gene therapy of inner ear dieases. Partâ…¢Protection of cochlea from aminoglycoside ototoxicity by Manganese superoxide dismutase gene in aging ratsObjective To determine the feasibility of manganese superoxide dismutase (MnSOD) gene therapy for protecting the inner ear against aminoglycoside- induced oxidative stress in aging rats. Methods The viral particles of recombinant adeno-associated viral vector 2-IRES-EGFP/MnSOD(rAAV2-IRES-EGFP/MnSOD) were injected into the perilymph through the round window membrane on the 6th week.To observe the feasibility of MnSOD gene therapy for protecting the cochlear function with the methods of Westernblot, ApopTag peroxidase apoptosis detection, MnSOD activity detection and effect on hearing. Results The apoptosis of cochlear tissue was partly reduced, the MnSOD activity is increased, and the hearing threshold is decreased after rAAV2-IRES-EGFP/MnSOD inner injection,compared with saline injection group. Conclusion MnSOD could play a partly role to treat oxidative damage of inner ear in aging rats.With the heredity baxkground of mtDNA 4834 bp common deletion, aminoglycoside ototoxicity can not be resist compeletly by antioxidant gene therapy.