The Role Of Small Molecules In Cochlea Hair Cell Regeneration

Sensorineural hearing loss influences the human being severely，it is alwayscaused by the death of hair cells in the cochlear. There are no effective clinicalmeans for curing it at present since the key point regeneration of hair cells inmammals is sill in research.Recently,molecular biology, molecular genetics andgenetic engineering have been taken into ear biology,which improved ourtechnology for inner ear investigation. Our research aim to study the role of smallmolecules in cochlear hair cell regeneration,which includs DMSO,DAPT andmiRNA.Part one: Cytotoxic effects of dimethyl sulphoxide on cochlear hair cells in vivo.OBJECTIVE: To assess cytotoxic effects of dimethyl sulphoxide (DMSO) oncochlear hair cells in vivo.METHODS: Healthy4-week-old SD rats (100-120g, n=40) of either sex wererandomly divided into four groups: artificial perilymph control group;0.1%DMSOgroup;1%DMSO group;5%DMSO group. The auditory brainstem response(ABR) test was carried out1day before drug administration and7days after drugadministration to determine the effects of drug administration on hearing.Using theconfocal microscope and HE staining，we observe the change of cochlear hair cells7days after drug administration. TUNEL staining was carried out at2hours，4hours，6hours and12hours after drug administration respectively in5%DMSOgroup.RESULTS: In the artificial perilymph control group and0.1%DMSO group, theABR threshold shift just a little at click,4kHz,8kHz,16kHz,and at32kHz thethreshold shifted below30dBSPL,with little or no damage in morphology. However,concentrations of1%caused considerable loss of OHC, when the concentration wasincreased to5%, the damage not only at outer hair cells but also at inner hair cells,and the ABR threshold shifted obviously.HE staining showed that supportingcells were affected also.TUNEL positive staining was observed in many types ofcells on the basilar memberane at2hours after drug administrtion.CONCLUSION: DMSO-induced damage was in dose-dependent.The hair cellsshowed various degrees of injury from the apical turn to the basal turn of thecochlea.DMSO destroyed not only hair cells,but also supporting cells.Cytotoxiceffects of DMSO was related to the apoptosis.Part Two: The effects of DAPT to hair cell regeneration in vivo.OBJECTIVE: To explore the effects of DAPT to hair cell regeneration in vivo.METHODS: DAPT was delivered into the right cochlea of rats between11-14daysold (n=15) through three ways，one is osmotic pump，the others are successiveadministration and intermittent administration through scala tympani.The confocalmicroscope was applied to observe the change of the basilar menbrane2weeks(theosmotic pump group) or1week(the other two)after the delivery.RESULTS: In the osmotic pump group atopic cells with both myosinVIIa andphalloidine positive were oberserved,but it happened in one ear only.In addition,inthe apical turn of two ears in the osmotic pump group,most hair bundles werelost,with scattered phalloidine staining on the top of the OHCs,besides,theorientation of steriocilia remaining was affected more or less.Hair cells andsupporting cells were seriously dameged in the other two groups,and there was nogeneration of new hair cells.CONCLUSION: DAPT delivery to the neonatal rat cochlea may have effects on thesteriocilia.Since the osmotic pump delivered drugs quantitatively and chronically,itshould be a better way to study the effcts of DAPT to the hair cell regeneration invivo.Part Three: Preliminary Study on the role of miRNA in the regeneration ofcochlear hair cellsOBJECTIVE: To investigate the differencs of miRNA expresion profile betweenthe new born rat and mature rat,hoping to find the role of miRNA in the regenerationof cochlear hair cells. METHODS: Microarray was used to explore the miRNA expresion profile of theP0rat(n=9) and P30rat(n=9).GO analysis was applied to analyze the main functionof the differential expression genes according to the Gene Ontology.Similarly,Pathway analysis was used to find out the significant pathway of the differentialgenes according to KEGG.RESULTS: There are16miRNAs increase in mature rat compared to that of thenew born rat. Two of the miRNA expression level were reduced in mature rat.Thehigh-enrichment GOs targeted by over-expressed miRNAs were involved in avariety of cellular processes.Functional analysis of miRNAs by KEGG revealed that19signal transduction pathways were upregulated and14were downregulated.CONCLUSION: The miRNA expression profile was different between new bornrat and mature rat.According to GO analysis and Pathway analysis,we found that thechange of miRNA expression was related to the maturation of the Corti,as well ascell function maintaining and the disability of hair cells proliferation.Furthermore,TGFβ signaling might play an important role in the hair cell regeneration.