| Normal hearing is crucial for communication, pleasure and awareness inhumans. A hearing deficit substantially affects social integration and quality oflife. The spiral ganglion neurons (SGNs) in the cochlea are the primary auditoryafferent neurons that transmit sound to the central auditory system. Variousototoxic treatments, heredity and the aging process can lead to the primary orsecondary degeneration of the SGNs, which leads to severe sensorineural hearingloss (SNHL). Furthermore, the loss of the SGNs reduces the effectiveness ofcochlear implants, which improve impaired hearing by directly stimulating theresidual SGNs in the absence of hair cells. However, in mammals, SGNs cannotregenerate from endogenous cells or be replaced by cell division. Therefore, therestoration of degenerated SGNs is important and essential for the treatment ofSNHL caused by SGN degeneration. Given the lack of endogenous regenerationand the limited therapeutic range, treatments based on the delivery of exogenouscells into the impaired cochlea offer new hope. To date, many studies haveaddressed this aspect of SNHL treatment; however, challenges still exist. Forexample, one challenge is to transplant cells into the inner ear without disturbingthe intracochlear structure and residual hearing. The ideal route for cells transplantation into the inner ear is to deliver the cells to the appropriate placewhere the transplanted cells can survive and migrate to damaged regions;moreover, further trauma to the homeostasis of the cochlea and residual hearingmust be minimized. Transplanting exogenous cells in the cochlea is difficultbecause the cochlea contains highly specialized, microscopic and complexstructures, and neither the perilymph nor the endolymph, which contain the scalavestibule (SV), the scala tympani (ST) and the scala media (SM), is suitable forcell survival. To address this problem, different techniques to transplant cells intothe mammalian cochleae have been reported in recent years. For example,intra-perilymph, intra-endolymph, into-modiolus and into-auditory nerve trunksurgical routes have been attempted; however, cells transplanted via theintra-perilymph and intra-endolymph routes have a low survival ratio, and themigration of the cells is also limited by the membranous and bony wall of thecochlear chamber. Other techniques, such as transplanting cells into the modiolusand auditory nerve trunk have resulted in a higher cell survival ratio; however,this technique runs the risk of serious hearing loss. In addition, surgery on theauditory nerve trunk can potentially cause severe intracranial infection.In this study, we established a rat SGN degeneration model, got GFP+NSCswhich obtained from the olfactory bulbs of C57BL/6-GFP mice at the embryonicday14.5, found a new route involving the transplantation of the neural stem cells(NSCs) into the cochlear lateral wall (CLW), a region between the lateral bonywall and the spiral ligament (SL). In addition, we assessed the damage caused bythe surgery and compared it with a method that delivers stem cells into the ST,which is believed to cause less trauma and is often used for transplanting cells,viruses, drugs and other substances into cochleae. We investigated if theCXCL12/CXCR4signal pathway has an important role of the migration anddistribution for transplanted NSCs.Part one: Procedures for the SGNdegeneration model [Objectives] To obtain the SGN degeneration model induced by ouabain forthe following experiment to transplant the stem cells into cochlea.[Methods] Of the sixteen rats used to detect the SGN degeneration model,eight underwent surgery and were treated with ouabain (ouabain octahydrate,O3125, Sigma). The remaining rats underwent surgery but were treated withnormal saline as a control. The left otic bulla of the rat was opened to expose thebasal turn of the cochlea and the round window niche.10μl of a10mM ouabainsolution in normal saline was placed in the RW niche for10min using a10μlsyringe. After injection, the pipe was removed, and the incisions were closed withsutures. The model was verified by cochlear sections, the auditory brainstemresponse (ABR) threshold and the hair cell count.[Results] H&E and β-Ⅲ-tubulin-stained sections exhibited only a fewsurviving SGNs in the SG of the basal turn at3d and7d after ouabain exposurewhen compared with the control profiles.Cochlear surface preparation revealedno missing hair cells in any of the three turns at7d after ouabain exposure. TheABR threshold in the the ouabain-treated groups were decreased at3d and7d.The results verified that the rat SGN degeneration model was established aftertreatment of the RW niche with ouabain in this study. This model is similar toprevious models reported for other animals [2,34], but it is the first modelreported in rats.[Conclusions] The results verified that the rat SGN degeneration modelwas established after treatment of the RW niche with ouabain in this study. Thismodel is similar to previous models reported for other animals [2,34], but it is thefirst model reported in rats.Part two: Cultivation of GFP+NSCs obtainedfrom the olfactory bulbs[Objectives]To obtain GFP+NSCs from the olfactory bulbs ofC57BL/6-GFP mice at embryonic day14.5for the transplantation of the rat cochlea.[Methods] The embryos were removed from their sacs. The embryonicbrains were removed, and the olfactory bulbs were dissected. The cells wereobtained by mechanical dissociation of the olfactory bulbs. After a mildtrypsinization, the cells were then incubated at37°C in a5%CO2atmosphere.Cells were passaged every4–7d the cells survived more than100passages inculture and maintained their stem cell characteristics. The majority of thecultured cells were GFP+NSCs, which were identified by the expression of GFPand nestin, a neural stem cell marker (mouse monoclonal nestin antibody,1:100,ab6124, Abcam Ltd., Hong Kong)(Fig.3B). The NSCs were harvested fortransplantation on passage20at a cell density of1×106cells/ml.[Results] The cells survived more than100passages in culture andmaintained their stem cell characteristics. The majority of the cultured cells wereGFP+NSCs, which were identified by the expression of GFP and nestin, a neuralstem cell marker The NSCs were harvested for transplantation on passage20at acell density of1×106cells/ml.[Conclusions] The results suggested that we have obtained the seed cells dfor cochlear transplantationPart three:Transplantation of NSCs into cochlea: throughlateral wall into the SG without invasion of ScalaMedia[Objectives] Because of the poor regeneration of the spiral ganglionneurons (SGNs) in the mammalian inner ear, the transplantation of stem cells hasbeen used to replace injured SGNs. However, the specific structures of themammalian cochlea form barriers for the migration of the exogenous stem cells.In this study, we attempted to transplant neural stem cells (NSCs) into the cochlear lateral wall (CLW) and estimated their migration to the spiral ganglion(SG) in a rat SGN degeneration model.[Methods] We transplanted the NSCs into the CLW and examined theirdistributions in the cochlea. In addition, we verified the precision of the injectionsite by using Fluorogold (FG) and assessed the damage caused by the surgery.[Results] We found that the procedure to transplant the stem cells into theCLW is convenient and that the injection site is precise. The NSC transplantedinto the CLW has a higher survival ratio, compared with previous studies thattransplanted embryonic stem cells into the scala tympani (ST), and migrate withsurprising efficiency to the SG in the SGN degeneration model. The distributionof the green fluorescent protein (GFP) positive cells suggested that the basilarmembrane (BM) is a potential channel that allows the transplanted NSCs to passfrom the spiral ligament (SL) to the SG. Although this method disrupts thefunction of the inner ear, the disruption is temporary, and the ear recovers in2weeks.[Conclusions] This study provides a novel method for inner eartransplantation that will contribute to the treatment of sensorineural hearing loss(SNHL) caused by SGN degeneration.Part FOUR:NSCs were regulated by CXCL12/CXCR4signalpathway after transplantation of cochlea[Objectives] To study on the regulation of the migration of the NSCs aftertransplantation into cochlea, which have damaged by ouabain[Methods] We investigated the expression of the CXCL12in the SGNdegeneration model reduced by applying ouabain to the round window niche(RW) of the cochlea; we examined the expression of the CXCL12/CXCR4in theNSCs, We examined if the area of the NSCs distributions in the cochlea have thehigh expressions of CXCL12simultaneously. [Results] We found that the NSCs express CXCL12/CXCR4, we verifiedthat the expression of the CXCL12in the SGN degeneration model is increasedin SG, BM, SV and SL, and we found that the area of the NSCs distributions inthe cochlea have the high expressions of CXCL12simultaneously.[Conclusions] This study verified that the migration of the NSCs wereregulated by CXCL12/CXCR4signal pathway after transplantation of cochlea... |
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