The Damage Of Transient Oxidative Stress In Rat Inner Ear And Protection

A lot of investigations show that oxidative damage is the latent cause of almost all diseases and it is also the main reason for noise induced hearing loss,sudden deafness ,presbycusis and drug induced deafness .the oxidative damage of inner ears is always the result of transient oxidative stress or transient oxidative stress for many times,so the transient oxidative stress applied in the investigation is more practical ;in the past most of the researches on the damage of hearing organ are about acoustic hair cells and the neurons of the spiral ganglion.in our research , the rat cochlear strial vascularies are the main target in order to explore the function of strial vascularies in fighting against the oxidative damage occur in inner ears. Investigation on the change of the cells' morphous ,function,and molecular biology during the transient oxidative stress may be helpful to find out the target to which the protectant should be applied in the future .At present , there is no effective medicine for deafness,most of the effective Chinese herb used to treat deafness is antioxidant ,so we used the powerful botanical antioxidant-the grape extractive (Procyanidolic Oligomers,OPC) to feed the rats ,and observe how OPC effect the retrogressive change of the rats's inner ears on morphous ,acoustic biology and biochemistryduring their natural aging process.For further investigation the mechanism of the grape extractive (Procyanidolic Oligomers,OPC) protecting inner ear from oxidative damage ,we take reverse transcription-ploymerase chain reaction technology to investigate how the transient oxidative stress and antioxidant effect the expression of Isk which is specific in strial vascularies ,and find out the function of strial vascularies in fighting against the cochlear oxidative damage .Providing the experimental base for further research of oxidative and antioxidant in inner ear. Methods1,drawing Corti's Organ and strial vascularies and culturing them in vitro7-day-old neonatal Wistar rats were used ,and they were decapitated.cut the head into two pieces and remove the brain .expose the bottom of the cochlea and take out of the integrated acoustic capsule under the anatomical microscope ,and put it into the physical solution ,and then remove the bone and expose spiral ligament .remove the strial vascularies from the media wall of the spiral ligament. Get the integrated Corti's Organ also .It takes 5 minutes to get them after the animals are sacrificed.the bioactivity of the strial vascularie and Corti' s Organ were not effected .Culture them in the bin with 5% CO2, 37℃.2,observing the vallate of Corti' S Organ and strial vasculariesThe strial vascularies of the experiment group ang the contral group were fixed and were stained by TRITC-Philloidin ,observe them under the upright fluoreascence microscope and take photographs.3,observing the nuclear of the strial vascularies cellsThe strial vascularies of the experiment group and the control group were stained by PI ,and the appearance of the nuclear and the morphological change of the nuclear under the upright fluoreascence microscope and take photographs.In order to identify the injured cells suffered the apoptosis , TUNEL Kit was used .The procedure was according to the note of the kit.4,the measurement of the ROS in strial vasculariesAfter the strial vascularies of the experiment group and the control group have been cultured for 0,10,30,60 minutes ,put DHR123 into the culture medium (final concentration is 20uM),observe the concentration of ROS according to the time course under LSCM(excitation:568nm,emission:615).The change of the concentration consists with the change of the fluoreascence intensity .Adobe Photoshop 7. 0 Software was used for quantitive analysis.5,the measurement of the mitochondria membrane potential of Corti' s Organ and strial vasculariesThe Corti' s Organ and strial vascularies in experiment group and control group were stained with JC-1 after being cultured for 30 minutes ,2 hours and 8 hours ,and were observed under the inversion fluoreascence microscope and take photographs.6,the measurement of ABR thresholdTest once before and after using of the medicine for 6 months.measure the ABR threshold value in the baffle electric screen booth,and record it .Use Danac-7stimulator acoustic to produce clicking sound , frequency:20 /s, scanner time: 10ms , smoothing :100-300Hz, the sod output range :0-100dB,signalsuperposition:128 times,and assessment the auditory threshold by Wave III threshold.7,the measurement of SOD in serum and cochlearMeasure the SOD in serum and cochlear after the animals had been dealt with the medicine for 6 months. Drawing cytocochleogram after ABR experiment ,take out of temporal bone ,and then get integrated basal membrane under anatomical microscope ,finally make them into basal membrane stretched preparation .they were stained by Phalloidin for 30 minutes ,and were observed under upright fluoreascence microscope and take photographs.8,investigate the expression of Isk mRNA of the strial vascularies marginal cells with RT-PCR.Extract the total RNA of the strial vascularies; Measure the OD value and determine the concentration and purity of the RNA; synthesizing cDNA by reverse transcription; Reverse transcriptase-ploymerase chain reaction (PCR) . Results1,the damage of the cells in strial vascularies and Corti' s Organ after transient oxidative stress(1) The TRITC-Philloidin stained strial vascularies in the experiment group show the celluar condense and dissolve after having been cultured for 2 hours or 8 hours. The strial vascularies cells in control group show negative PI stain after having been cultured for 8 hours. The PI stained strial vascularies in the experiment group show positive PI stain after having been cultured for 2 hours or 8 hours, and the nuclear changed.in order to test the way they died, use TUNEL Kit to observe, the result show part of the died cells suffered apoptosis.(2) The hair cells in the control group treated with TRITC-Philloidin line up in order without lost hair cells after having been cultured for 8 hours ,but some of the hair cells in the test group lost after their having been cultured for 8 hours .2,the change of the mitochondria membrane potential in the strial vascularies and Corti' s Organ after transient oxidative stress(1) The change of the mitochondria membrane potential in the strial vascularies: the JC-1 treated strial vasculary in control group show the intense red or orange colored fluorescence after 8 hour-culture. This suggest the exist of mitochondria membrane potential .but the JC-1 treated strial vasculary in control group showed the red ,yellow or green after having been cultured for 30 minutes ,2 hours or 8 hours respectively. This suggest that the mitochondria membrane potential in these cells disappeared continuously.(2) The change of the mitochondria membrane potential in Corti' s Organ : after transient oxidative stress: the Corti' s Organ in the control group which were treated with JC-1 shows the mitochondria membrane potential after been cultured for 8 hours , and centered in the area of cover membrane and hair cells .However the mitochondria membrane potential of the Corti' s Organ disappeared after having been cultured for 2 hours .3,the measurement of the ROS in strial vascularies after trasient oxidative stressAfter the strial vascularies of the experiment group and the control group have been cultured for 0,10,30,60 minutes, put DHR123 into the culture medium (final concentration is 20uM), observe the concentration of ROS according to the time course under LSCM(excitation:568nm,emission:615).the change of the concentration consists with the change of the fluoreascence intensity .There is no ROS in the control group ,the strial vascularies in experiment group showed weak fluorescent light after cultured for 10 minutes ,the light is slightly reinforced after 30 minutes ,and obviously reinforced after 60 minutes ,the intensity of the tissue cultured for 120 minutes is similar to that of 60 minutes.4,the measurement of ABR threshold and SOD in vivo(1) ABR threshold :there is no significant difference between the threshold of the two groups before OPC was used : P>0. 05. There is the significant different between the ones of the two groups after OPC was used :P<0. 01.(2) The SOD in the serum and the cochlear: Compared with the control group ,the activity of the SOD in the OPC-treated group from the serum and cochlear was reinforced.(3) The lost of the outer hair cells in the cochlear basal membrane:the hair cells of the middle turn hardly show hair cell lose ,the hair cell in the top turn of both groups show hair cell lose ,but there is no significant different .For the bottom turn , the percentage of the lost hair cells in senile group is 14%, while that in OPC-treated group is 7%.5,the measurement of the expression of Isk mRNA in strial vascularies with RT-PCRThe result of RT-PCR:there is aspecial amplification band for Isk mRNA at 368bp.Compared to the intensity of the amplification band in control group, the intensity is weak after H₂O₂ has been used .treatment of H₂O₂ + OPC, the weaken intensity was improved.Conclusions1,Transient oxidative stress can stimulate cochlear produces ROS spontaneously, the integrity of the mitochondrial membrane is destroyed , lead to the injure and death of the Corti' s Organ and strial vascularies .2,Applying the grape extractive (OPC) to feed the senile rats in long-term can delay the hearing decline.3,Transient oxidative stress can attenuate the expression of the special Isk mRNA ,natural botanical antioxidant-grape extractive (OPC) can restore the expression of Isk mRNA which is injured by oxidation ,and protect strial vascularies in cochlear.