Comparison Of Neural Stem Cells Transplantation Into Cochlea Via Roundwindow Membrane With The Basal Turn Of Cochleostomy

BACKGROUND AND PURPOSES: SNHL affects the health of individuals seriously.Loss and degeneration of HCs and SGNs always lead to hearing loss or deafness. In the past,people believed that loss and degenerateion of mammalian auditory HCs and SGNs were irreversible.Recent studies on NSCs transplantation into the inner ear suggest that NSCs give hopes for treatment of damaged HCs and SGNs.Malgrange,et al found Nestin (+) (mark of NSC)cells in the cochlear tissue during the late embryonic and newborn rat.Mao,et al reported that NSCs are located in scalae vestibule,scalae tympani, spiral ganglan and stria vascularis,and on 14th day after transplanted into the cochlea.Ito,et al found that many surviving transplanted-NSCs were in the organ of Corti on 28th day after transplantation,some of them took on the distinct morphology of inner or outer HCs. Iguchi,et al reported that NSC-derived cells survive in the perilymphatic space in the cochlea for at least 28 days,and could express neurotrophins BDNF,which not only have efficacy for protection of HCs and SGNs from various toxic insults,but also enhance the sensitivity,generation and functional maintenance of SGNs.For future human application , it is necessary to develop a NSCs delivery method that preserves hearing function as well as cochlear architecture.In this regard,the routes of deliving NSCs into the cochlea is a key element of success in cell therapy.Up to date,several possible methods have been used inforeign materials transplantation into cochlea:?microinjection into the scala tympani via RWM;(2)microinjection into the scala tympani through the basal turn of cochleostomy;? injection into the endolymphatic sac accessed from the posterior cranial fossa;@injection through semicircular canals;?injection through the CSF.That via the RWM and through the basal turn of cochleostomy are the mostly possible approaches for NSCs transplantation into cochlea.The experient is that transplantation NSCs into cochlea via the RWM or through the basal turn of cochleostomy,evaluate hearing loss and HCs damagement or not after operation.Compare the numbers of survival transplanted-NSCs in the scalae tympani between different groups on 7th after transplantation by Brdu immunocytochemical staining,and determine which of the two is more effective.Identified the express of BDNF in the scalae tympani,compare before NSCs injection with after NSCs injection,in order to evalue the ability of expression of NSCs.MATERIALS AND METHODS: The experient consists of two parts.The first part is CELL EXPERIENTjNSCs are isolation from newborn guinea pig hippocampus tissues, some identified with Nestin immunocytochemical staining,some induced to differentiate by FBS,some labeled with Brdu.The second part is ANIMAL EXPERIENT,Sixty-eight guinea pigs,either male or femalehealthy,sensitive in preyer reflex,no signs of middle ear infection,the baseline click-ABR thresholds of 20~30dBSPL,weighing from 200 to 300 grams,are randomly divided into 7 groups.Group I (4 guinea pig, 8 ears),the normal control group,were under no treatment,as for the normal contrast of test the baseline click-ABR thresholds,stread flake and NBT staining of cochlea fundus membrane.Group II (20 guinea pig, 40 ears),only making a hole in base turn of cochleostomy,no deafened and no NSCs injection,every 4 guinea pigs are sacrificed respectively on 3rd,7th,14th,28th day after operation,stread flake and NBT staining of cochlea fundus membrane,compared with groupIII;the left 4 guinea pigs are sacrificed respectively on 7th day after operation, BDNF immunocytochemical staining of scalae tympani,compared with groupIV.GroupIII (20 guinea pig, 40 ears),only making a hole in RWM,no deafened and no NSCs injection, every 4 guinea pigs are sacrificed respectively on 3rd,7th,14th,28th day after operation, stread flake and NBT staining of cochlea fundus membrane,compared with group II ;theleft 4 guinea pigs are sacrificed respectively on 7th day after operation,BDNF immunocytochemical staining of scalae tympani,compared with group V.GroupIV (8 guinea pig, 16 ears),Brdu-labeled-NSCs injection through cochleostomy, no deafened,4 guinea pigs are sacrificed respectively on 7th day after operation,paraffin slice Brdu immunocytochemical staining of scalae tympani,compared with groupVI&V;the other 4 guinea pigs are sacrificed respectively on 7th day after operation,BDNF immunocytochemical staining of scalae tympani,compared with group II .Group V (8 guinea pig,16 ears),Brdu-labeled-NSCs injection through RWM,no deafened,4 guinea pigs are sacrificed respectively on 7th day after operation,paraffin slice Brdu immunocytochemical staining of scalae tympani, compared with group IV &VII ;the other 4 guinea pigs are sacrificed respectively on 7th day after operation,BDNF immunocytchemical staining of scalae tympani,compared with groupIII.GroupVI(4 guinea pig, 8 ears),Brdu-labeled-NSCs injection through cochleostomy in deafen guinea pig,4 guinea pigs are sacrificed respectively on 7th day after operation,paraffin slice Brdu immunocytochemical staining of scalae tympani,compared with groupIV&VlI.GroupVn(4 guinea pig,8 ears),Brdu-labeled-NSCs injection through RWM in deafen guinea pig,4 guinea pigs are sacrificed respectively on 7th day after operation,paraffin slice Brdu immunocytochemical staining of scalae tympani,compared with groupVI& V .The data were disposed by software SPSS12.0.RESULTS: 1 Nest-like clusters of NSCs are obtained in the suspension,and nestin immunocytochemical staining is brown.2 NSCs are induced to differentiate by FBS, and the differentiated cells have 1~2 axon or several dendrite.3 The maximum average ABR threshold shift after operation is in the range of 10~15dBSPL in group II and III,and it can turn to normal in 2 weeks.4 Either in groupIVor in groupV,there are numbers of BDNF stainings in the basal turn of scalae tympani on 7th after operation,but none of in group II orIII.5 Number of Brdu stainings in scalae tympani of groupIVis more than of group Von 7th after operation. 6 Number of Brdu stainings in scalae tympani of group VI is more than of groupVDon 7th after operation.7 Number of Brdu stainings in scalae tympani of groupVIis more than of groupIVon 7th after operation.8 Number of Brdu stainings in scalae tympani of groupVD is more than of group V on 7th after operation .CONCLUSIONS:1 NSCs can be isolated from newborn guinea pig hippocampus tissues,identified with Nestin immunocytochemical staining.2 Transplantation-derived NSCs or its' differentiated cells can express BDNF.3 In the groupe of undeafened guinea pigsneither NSCs injection through the basal turn of cochleostomy nor through the RWM have a significant effect on the click-ABR threshold measured on 14th after NSCs transplantation.4 NSCs inject through the the basal turn of cochleostomy are more efficient than via the RWM in hearing,as well as deafen guinea pigs.5 Either through the the basal turn of cochleostomy or through the RWM,surviving NSCs in scalae tympani of deafened guinea pigs shows more numbers than of undeafened ones.