Study Of AD-NT3 Gene Therapy For Auditory Neurons Reservation Within The Cochlea Of Guinea Pig Following Intensive Stable Noise Trauma

Numerous studies have shown that neurotrophic factors(NTFs) are closely connected with mammalian peripherial auditory system . NTFs not only play an important role in the regulation of development , maturity, prolifaction, and differenation of neurosensory epithelia and auditory neurons, but also have strongly ability to maintain the specific function in adulthood and prevent or reduce secondary degeneration after the destruction of auditory hair cells. Neurotrophin-3 (NT-3) is a member of the family of growth factors, whose mRNA presented mainly in the organ of Corti. TrkC , the high affinity receptor of NT3, has its mRNA presented in spiral ganglion neurons greatly. A variety of studies in vitro and in vivo suggest NT-3 to be the primary survival factor for the inner ear and likely a potential therapeutic agent for sensorineural deafness (SN'D). However, delivery of polypeptide growth factors to the cochlea is difficult because their plasmatic half-life are very short and presence of the blood-labyrinth barrier. Gene therapy(GT) may overcome these problems by locally producing the desired therapeutic protein in a given cochlea. Adenovirus is currently one of the most appropriate gene therapy vectors for its properties. Now, genetic engineering technique can successfully constructed replication-defective adenovirus vector carrying NT-3 cDNA(Ad-NT3) and it provide a new method to resolve the problem. In order to further investigate the effects of intra-cochlea gene therapy on the restoration or repair of the damaged primary' auditory neurons, a postnatal mammalian animal model were made by intensive stable noise exposing and the gene therapy procedure within cochlea were established. By using immunohistochemistry, histopathology, ultrastructural pathology and auditory electrophysiology techniques, weobserved the morphological and functional changes after noise trauma and evaluated the safety and feasibility of Ad-NT3 introduction into the cochlea of guinea pigs. Through these methods, we also assessed the biological activities of Ad-NT3 gene transduction within cochlea and its rescuer effects on the noise-injured auditory neurons. Based on the results of these series experiments , the primary conclusions were made as follow:1. A peripherial target-deprived animal modal can be achieved by the intensive stable noise damage. The irreversible acoustic injury from exposing to a intensive steady noise(130 dB SPL x 5 hours) were obtained in the guinea pigs. The pathological changes in the auditory nerve consist of primary impairment in neurosensory epithelia of organ of Corti and secondary retrograde degeneration in the neural fibers which extending to spiral ganglion neurons. The neurofilament protein immunohistochemistry reaction also confirmed the degenerative procedure following noise trauma. At the level of exposure intensity of 130 dB, a severe CAP and ABR alternation are found, both in threshold and latency. Such drastic damage lead principally to a near completely loss of auditory epithelia and permant threshold shift. The pathological changes were consistent with auditory electrophysiological alternations.2.The feasible and safe procedure for gene therapy within cochlea were also explored. By drilling a small hole on the bony wall of tympanic scala in the basal turn following cochleostomy, a total of 5 n 1 Ad-NT3 was inoculated into the perilymphatic space of guinea pig cochlea. The pathologic and electrophysiological examination after injection procedure showed no significant structure changes and hearing loss after operation. Our results suggested it is reasonable and safe to perform the gene transduction into inner ear.3.To determain whether the introduced Ad-NT3 would affect cochlea, paraffin-embedded samples and ABR were examined in animal model. In serial sections, neither cellular degeneration of middle ear nor deviation of cochlea tissue was observed in the 7th days after noise exposing. The infection of replication-defective adenovirus itself has no side-effect to cochlear stru...