| Neurotrophins such NT3 and BDNF and protease inhibitors such as calpastatin are known to enhance the survival of neurons; however, delivery of these neuroprotective agents into the inner ear is problematic because of their large size and the need for targeted delivery of these compounds. Adenovirus mediated gene transfer represents a new approach for targeted delivery of NT3, BDNF and calpastatin into inner ear to enhance spiral ganglion neurons (SGNs) survival following hair cell damage. To determine if adenovirus mediated gene transfer of NT3, BDNF and calpastatin could enhance the survival of SGNs, we developed an organotypic model of delayed SGN degeneration by initially destroying the hair cells with 2 mM gentamicin. The percentage of surviving SGNs decreased from approximately 87% at 1 day post-gentamicin to around 5% at 14 days post-treatment. To rescue SGNs from gentamicin damage, recombinant adenoviruses for NT-3, BDNF and EGFP were constructed using pAdTrack-CMV and pAd easy-1 systems. NT3, and BDNF genes were amplified by PCR, inserted into GEM-T vector, and the resulting recombinant vectors inserted into pAdTrack-CMV containing EGFP. Recombinant plasmids were cotransformed into E. coli and kanamycin resistant recombinant plasmids were transfected into 293 cells for production of recombinant adenoviruses expressing NT-3 or BDNF. AdV-NT3 (6.9×109 VP/ml) and AdV-BDNF (6×109 VP/ml) were transfected into rabbit bone marrow cells and western blots carried out to confirm the expression of NT3 or BDNF. To determine the number and location of cells infected by the recombinant virus, AdV-EGFP, normal and damaged cochlear cultures were treated with different concentrations of AdV-EGFP. In normal cochlear cultures, many EGFP transfected cells were seen in the outer sulcus and interdental cell region; few EGFP positive cells were seen in the hair cell, inner sulcus or spiral ganglion region. In cochlear cultures damaged with 2 mM gentamicin, there was an overall increase in the number of EGFP labeled cells in the Deiters' cell region and the interdental cells region. Pretreatment of cochlear cultures with collagenase resulted in an increase in the number of EGFP labeled cells in the outer sulcus and hair cell regions. These results indicate that the adenovirus preferentially transfects supporting cells in cochlear cultures. To determine if AdV/EGFP-NT3 and AdV/EGFP-BDNF would promote the survival of SGNs, cochlear cultures were pretreated for 3 h with 109 VP/ml AdV/EGFP-NT3, AdV/EGFP-BDNF or the combination; afterwards the samples were treated for 24 h with 2 mM gentamicin and harvested 7 days later. Only 15% of SGN were present 7 days after the gentamicin treatment; pretreatment with AdV/EGFP + BDNF, AdV/EGFP+NT3 or the combination significantly increase SGN survival to 39%, 51% and 69% respectively. These results indicate that BDNF, NT3 or the combination can significantly enhance SGN survival after gentamicin-induced hair cell death. Although NT3 and BDNF promote the survival of SGNs, they failed to rescue 100% SGNs suggesting that gentamicin might induce SGN loss by some other pathway. Calpains, calcium activated proteases, have been implicated in gentamicin ototoxicity. To determine if calpastatin, a calpain inhibitor, would prevent SGN death, cochlear cultures were pretreated for 3 h with AdV-calpastatin (109 VP/ml), AdV-NT3, AdV-BDNF or various combinations followed by gentamicin (2 mM, 3 h). AdV-calpastatin significantly increased SGN survival in cochlear cultures treated with gentamicin. SGN counts per 0.06 mm2 were 45 for AdV-NT3, 34 for AdV-BDNF, 38 for AdV-calpastatin, 45 for AdV-BDNF combined with AdV-calpastatin and 55 for AdV-NT3 combined with AdV-calpastatin. The results suggest that calpains could play a role on SGNs delayed death. Our results indicate recombinant adenoviruses mainly transfect the supporting cells in cochlear cultures. Cochlear cultures pretreated with AdV-BDNF, AdV-NT3 and AdV-calpastatin significantly enhance t...
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