Experimental Studies On Protection Of Nerve Growth Factor To Spiral Ganglion Cells

Most forms of deafness result from irreversible loss of cochlear HCs and lead to subsequent apoptosis and degeneration of the SGNs and auditory nerve. Less frequently, patients with auditory neuropathy suffer hearing loss due to primary degeneration of dysfunction of the auditory nerve. By providing direct electrical stimulation to the SGNs, cochlear implants effectively replace the mechanosensory transduction function of lost HCs and provide the user with substantial hearing benefit. Currently, most patients retain sufficient SGNs to benefit from CIs. The success of CIs depends , in part, on the number of normal SGNs. Thus, while as few as 10% of SGNs are sufficient for success with current CIs, SGN degeneration may prove to be a limiting factor for optimal outcomes with more advanced CIs. Thus, strategies aimed at preserving or regenerating deafferented SGNs carry critical implication for further advances. The survival of SGNs depends on the neurotrophic factors secreting from Corti's organ,cochlear nuclear and nerve fibers etc. Besides, the neurotransmitter such as glumatic-acid,SP and ACH coming from HCs also have effects on the survival of SGNs.NGF has important effects on the survival and regeneration of SGNs , growth and molding of axon and building of neurol network. NGF is a high efficient and multipotent neurotrophic factor. Three subunits which areα,β,γcompose the whole molecule and arranges according toα2βγ2. Among them, only theβsubunit is the functional one. The molecular weight is 23KD. During the development of nerve system, NGF can promote the differentiation and control the number of neurons. In mature periods, NGF can maintain the functions of sensory neurons and CNS and promote the branching of axis cylinder. When recovering from damage, NGF can support the survival of neurons and the growth of nerve fibers. Lefebvre found acoustic vesicle could secret a kind of factors similar to NGF when adding E12 acoustic vesicle to rabbit CVG. The factor could promote the survival of the neurons and the occurrence of the axon in vitro. The NGF-antibody could block up the function.Only after NGF binds with its receptors can it play the roles. The high affinity receptor TrkA has been well known that it is the functional receptor of NGF. After binding with NGF, TrkA wakes the activity of lu-PKase and a series of cascade reaction are occurring, and then the actions of maintaining survival and promoting development are generating. Dai had founded that TrkA distributed in the SGNs of mouse and rabbit's cochleae. The experiment hinted that NGF might bind with its receptors and had the effects on SGNs.At present there are many difficulties such as the obtaining and purification of exogenous NGF and limitation of resources. Because of giant molecular weight, NGF can't move across the blood-labyrinth barrier. Cochlea is coated in osseous labyrinth and forms an relative independent microenviroment because of blood-labyrinth barrier. As membranous labyrinth is hunging in the liquid environment composed with endolymph and perilymph ,exogenous substances have the opportunity of diffusion through cochlea. The characteristic give the possibility of gene transferring into the inner ear. Adenovirus belongs to linearity double strands DNA virus. It can infect all kinds of cells including neurons. The features have the special sense for correcting the gene defect of NS. As for efficiency and safty, adenovirus has becoming an useful carrier in gene engineering.NGF gene plays key roles in the differentiation and development of neurons. It is also indispensable to maintaining survival and promoting regenetation of mature NS. To study the protection of NGF to SGNs, we have detected the distribution of NGF protein during three development periods in cochlea first, then built a recombinated adenovirus vector with enhance green fluorescence protein report gene and NGF gene, AD-NGF, and the virus was infected into SGNs in culturing, at last, the virus was transfected into cochlea in vivo, the structures of SGNs and acoustic function were detected. The main results were as follows:1.The paraffin section of three development period(E20,P7,adult) were prepaired and the samples were stained by IHC for detectingβNGF protein distribution in cochlea in first part of our research. It was found that in E20 the ectoloph part (differentiated to Corti's organ) and immature SGNs were dyed. In P7 and adult cochleae, the structures had developed and SGNs and its axones had masculine dyeing. HCs in basal membrane were also dyed. The quantity ofβNGF protein were measured by WB. It would get a result that the quantity was most in P7 and lest in adult period among three periods.2. Extracted total RNA from mouse glandulae maxillaries, 924bp fragmentβNGF cDNA was amplified by RT-PCR. The fragment was recovered and purified to recombinate with a adenoviral shuttle plasmid pAdtrack-CMV carried eGFP report gene to build a new recombinant plasmid pAdtrack-CMV-eEGFP-βNGF, E.coli DH5αwere then transfected by the new plasmid .Expanded recombinant plasmids were extracted from transformed DH5αto identify by endonuclease-assay and electrophoresis and then the plasmid was homologous recombinated with adenoviral bone protein pAdeasy-1. The whole adenoviral expressing carrier was recombinate and was identified by electrophoresis and DNA sequencing. It was confirmed that 924bpβNGF cDNA fragment was inserted into the right site of the vector correctly. The DNA sequence was coincidence with that in GenBank database by comparision-assay. The correct recombinant plasmid was transfected into 293 cells by liposome transfection. The mature viruses would be packaged in 293 cells. After transfecting, the weak green fluorescence could be seen under fluorescence microscope on the second day. More days went, more and higher strength fluorescence could be observed. During 7～10days after transfection, the numbers of cells eruptiNGF luorescence were the most and the strength of lights were the highest. The morphouses of 293 cells appeared bead-like. After that, the fluorescence was weak. It would be an appropriate period for gathering virus at this time. After amplification virus, the production was abundant. The titre of virus was 1×107.4PFU/mL by TCID50 method. The virus obtainedβNGF cDNA fragment was detected by PCR and WB methods.3. Abstraction SGNs from mouse(P3～5) under anatomical microscope.The neurons were cultured in plates spreading 0.1% Polylysine. Ater 12h, the cells began to adhere and 24h later began to extend. The morphous of cells and length of axon were normal in 10 days . From then on the cells began to disfiguration. On the second day after culturing, virus was added into the culture liquid. The weak green fluorescence could be watched on the second day after transfection. More days went, more and higher strength fluorescence could be observed. On the fifth days after transfection, the numbers of cells erupting fluorescence were the most and the strength of lights were the highest. The time could last for 14days. The result showed that the virus could infect SGNs and the report gene could transcrip successfully.The remaining numbers of normal SGNs were much more than those untransfected.4. For the purpose of detecting the protection ofβNGF to basal-membrane,we trans- fected virus into basal-membrane cultured in three diamensions. It was showed that the whole basal-membrane sent out green fluorescence besides SGNs, and on the seventh day the density and the intensity of fluorescence are the most. The distribution concentrated in HCs and SGNs. After cultured 14 days, the orderliness in the basal-membrane was much more better than that untransfected.5. To study the function ofβNGF for cochlea and hearing.Guinea pigs were divided into four groups: A virus group,B GM group,C virus+GM group and D normal group. By round window membrane injection, the virus was injected into the cochlea. In the operation, temporal muscular fasciae was used to stopping leakage of virus from cochlea.we established a hearing-loss guinea-pig model by gentamicin (GM) intramuscular injection. In C group, we imported virus into cochlea 2 days before GM injection. TheβNGF function was confirmed by acoustic brain-stem evoked potentials (ABR) and SGNs TB staining. Results showed that the morphous of SGNs were almost normal in C group, but the ABR hearing threshold had significant difference to the normal ones. In this study, we got messages ofβNGF distribution during three development periods through IHC and WB. The result was a foundation for continuing study that the recombinant adenovirus could infected SGNs and expressβNGF protein. The adenovirus could infect the whole cochlea and had no obvious harmness. So the carrier would have possibility to apply in cochlear gene engineering.βNGF could protect SGNs from GM damage and could be imported into cochlea by adenovirus successfully. The study would supply a kind of method for deaf prevention and treatment.