An Experimental Investigation On Dexamethasone Antagonizes The Acoustic Trauma And The Ototoxicity Of Gentamicin

Noise and drugs induced heating loss as neurosensory deafness influence our quality of life severely.It is a long-term topic for many otologists to devote themrselves to investigate the prevention and cure of acoustic trauma and ototoxicity of gentamicin.Dexamethasone,as a synthetical glucocorticoid,has lots of physiological functions.It is reported that dexamethasone attenuates ototoxicity of noise and aminoglycosides drugs,however,its mechanisms are known little.Dexamethasone,as a kind of glucocorticoid,produces a marked effect through binding with glucocorticoid receptors located in the kytoplasm.It is essential to comprehend the distribution of glucocorticoid receptor(GR) in cochlea.Investigative methods including ELISA,in-situ hybridization,immunohistochernistry in previous studies.Few literatures can be fiound on the distribution of GR in cochlea and effects due to the acoustic trauma.Notch signaling is involved in proliferation differentiation of almost all cells.Hes1(hairy and enhancer of split 1) is its effective agent.The elevation of Hes1 mRNA level suggests the activation of Notch signaling,leading to gathering of CDKI p21^Wafl/Cip,which is closely related to the cell apoptosis.The relationship among the acoustic trauma,dexamethasone and Hes1 mRNA level can be one of mechanisms of dexamethasone antagonizing the acoustic trauma.However,little is known about it.It is reported dexamethasone antagonizes AmAn ototoxicity in vivo.Experiments in vitro avoid the systemic interferences,which make the investigative route more convenient and reliable.However,few literatures can be found on whether dexamethasone antagonizes ototoxicity of gentamicin in vitro.One of possible mechanisms for AmAn ototoxicity is the inhibition of calcium-sensitive outward potassium current of the outer hair cells.Few literatures can be found on whether the ion channel mechanism is involved in dexamethasone antagoning ototoxicity of gentamicin.The present study is designsd to explore the effects of dexamethasone on the acoustic trauma and the ototoxicity of gentamicin,and the possible underlying mechanisms.Immunofluorescent technique fixing,and fluorescence intensity semiquantitative analysis are detected for studying the distribution of GR in cochlea of guinea pig and the effects due to the acoustic trauma.Auditory brainstem response(ABR) and changes in cochlear morphology are detected for studying the effect of dexamethasone attenuate the acoustic trauma in cochlea.RT-PCR and real time PCR technique are used to discuss the effects of the acoustic trauma and dexamethasone on hes1 mRNA level in cochlea of guinea pig.Immunofluorescent technique fixing and changes in cochlear morphology are detected for studying the effect of dexamethasone attenuates the ototoxicity of gentamicin in vitro.Auditory brainstem response(ABR) is detected for studying the effect of dexamethasone attenuating the ototoxicity of gentamicin in vivo.The Whole cell patch clamp technique is utilized to investigate ion channel mechanism of ototoxicity of gentamicin and dexamethasone antagoning it.PartⅠThe Experimental Investigation on Dexamethasone Antagonizes the Acoustic Trauma1.The distribution of GR in cochlea of guinea pig and effects due to the acoustic trauma.Purpose To explore the distribution of GR in cochlea of guinea pig and effects due to the acoustic trauma.Methods White guinea pigs are randomly divided into 2 groups:experimental group and control group.Noise exposure(white noise,115dB SPL,3h) is exerted in experimental group.Decapitation and cochlear separation are done after 2h from the noise exposure termination.Empty treatments are done in the control group. Then,frozen sections are prepare.Immunofluorescent technique fixing,and fluorescence intensity semiquantitative analysis are detected for studying the distribution of GR in cochlea of guinea pig and the effects due to the acoustic trauma.Results(1) The fundamental positive positions of GR are located in spiral ligament,stria vascularis,spiral limbus,organ of Corti and spiral ganglions.For fluorescence intensity of GR,there are no significant difference in spiral ligament, stria vascularis,spiral limbus iand spiral ganglions(P＞0.05).Hower,there are significant difference between these positions and organ of Corti(P＜0.05).(2) The fluorescence intensities of GR located in spiral limbus,organ of Corti and spiral ganglions are similar in noise exposure group and control group(P＞0.05).The fluorescence intensities of GR in spiral ligament,stria vascularis are much lower in noise exposure group(P＜0.01).Conclusion The distribution of GR in cochlea of guinea pig are located in spiral ligament,stria vascularis,spiral limbus,organ of Corti and spiral ganglions.The highest and least position are lateral wall and organ of Corti,respectively.Acoustic trauma can decrease GR expression in all positions of cochlea,particularly in spiral ligament and stria vascularis.2.Dexamethasone attenuates the acoustic trauma via down-regulating cochlear hes1 mRNA levelPurpose To explore that dexamethasone attenuates the acoustic trauma and the relationship with hes1 mRNA level in cochlea.Methods Based on the preliminary experiment,guinea pigs are randomly divided into 5 groups:control group(NS ip qd,vegetable oil im qd),noise group(NS ip qd,vegetable oil im qd and noise exposure),dex+noise group(Dex ip qd,vegetable oil im qd and noise exposure),RU486+noise group(NS ip qd,RU486 im qd and noise exposure),dex+RU486+noise group(Dex ip qd,RU486 im qd and noise exposure). The dose and concentration of dexamethasone are 1mg/kg,0.5mg/ml,and that of RU486 are 20mg/kg,25mg/ml.All drugs are administered once a day for consecutive 5d.Animals are exposed to white noise(115dB SPL,3h).Immediately after the animals are sactificed,the cochlea are removed and made into tissue homogenate.The hes1 mRNA expression in cochlea is determined by quantitive realtime-PCR.Hearing thresholds of auditory brainstem responses(ABR_S) of experimental animals are measured before and 24h after noise exposure.For control group,ABR_s are measured in initiation and end of experiment.The NBT staining and basal membrane stretched preparation are practiced in all groups.Results(1) White noise(115dB SPL,3h) makes the cochlear hes1 mRNA level to 3 times compared with the control group.Dexamethasone pretreatment for 5d makes the cochlear hes1 mRNA level close to that of control group.The cochlear hes1 mRNA level of dex+RU486+noise group is similar with that of noise group,which suggest that RU486 almost blocks the effect that dexamethasone down-regulates cochlear hes1 mRNA level.(2) The mean threshold shifts of ABR of control group,noise group,dex+noise group,RU486+noise group and dex+RU486+noise group are 0.5 dB,29.56dB,11.46dB,26.93 dB,27.38dB after click and 0.5～1.40dB,32.75～34.78dB,12.75～15.85dB,30.15～33.26dB 30.26～33.52dB after short tone,respectively.There are significant differences for the mean threshold shifts of ABR in noise group,dex+RU486+noise group compared with those of dex+noise group(p＜0.05).(3) The cochlear surface preparation showed that the damage in noise group is severe but very slight in dex+noise group.Conclusion Dexamethasone has a potential protective role against the acoustic trauma possibly by down-regulating cochlear hes1 mRNA level.PartⅡThe Experimental Investigation on Dexamethasone Antagonizes the Ototoxicity of Gentamicin1.Dexamethasone attenuates the gentamycin-induced ototoxicity in vitroPurpose To explore whether dexamethasone attenuates the gentamycin-induced ototoxicity in vitro.Methods Based on successful establishment of organ Corti's organ cultural system,the p3 SD rat are randomly divided into 3 groups:Control group,GM group and Dex+GM group.Corti's organs are cultured for 72h.Rhodamine-phalloidin staining and immunofluorescent technique are used for observing growing status of Corti's organs.Results No significant hair calls deletion is found in control group.Significant OHC deletion is found in GM group,while no obvious change in IHC.OHC deletion degree is better in Dex+GM group,while no obvious change in IHC.Conclusion Pretreatment of dexamethasone attenuates the gentamycin-induced ototoxicity in vitro.2.Electrophysiologic studies for flexamethasone attenuates the gentamycin-induced ototoxicityPurpose To explore ion channel mechanism of dexamethasone attenuates the gentamycin-induced ototoxicity.Methods(1)Guinea pigs are randomly divided into 6 groups:control group(NS 0.5ml,ip),GM group(GM 80mg/kg,im),Dex-1 group(Dex 0.2mg/kg ip),Dex-h group(Dex 1mg/kg ip),Dex-l+GM group(Dex 0.2mg/kg ip,GM 80mg/kg,im) and Dex-h+GM group(Dex 1mg/kg ip,GM 80mg/kg,im).Before and after all drugs are adminietersd,ABRs is measured,and the threshold shifts are calculated afterward.(2) The whole-cell patch clamp techniques are used to study the effects of gentamycin(0.1,1,10,100,1000umol/L) and dexamethasone(0.1,1,10,100, 1000umol/L) on calcium-sensitive outward potassium currents in the isolated outer hair cells of guinea pig cochlea.Results(1)The mean threshold shifts of ABR are 1.0-1.6dB in control group,Dex-l group and Dex-h group,10.6dB of click and 5.0-21.0dB of tone in GM group with the most prominent at 6,8KHz stimuli.For Dex-l+GM and Dex-h+GM groups,the mean threshold shifts are 5.8-7.2dB after click,2.5-5.2dB after short tone with the values of click and 6,8KHz significantly lower than those in GM group (p＜0.05).(2) Gentamycin reduces calcium-sensitive outward potassium currents in a concentration dependent manner,while dexamethasone increases it as same manner as gentamycin.Conclusion Dexamethasone can remarkably attenuates the gentamycin-induced ototoxicity,and has no obvious effect on auditory function.Gentamycin reduces calcium-sensitive outward potassium currents in OHC,which suggests that is one of the mechanisms of acute ototoxicity.The possible mechanism of Dexamethasone antagonizing the ototoxicity of gentamicin is increased calcium-sensitive outward potassium currents in OHC.