Study Of Sodium Salicylate On The Plasticity Of Ribbon Synapse And Auditory Cortex Of Auditory Pathway In Rats With Tinnitus

Objective:Tinnitus is a common disease with high incidence,the treatment result is poor.The investigation shows that 6%-10%adult with chronic tinnitus,about 1%-3%of the people with tinnitus seriously affect the normal work and life,and become one of the urgent problems in clinic.At present,there are two hypotheses about the pathogenesis of tinnitus:one is that tinnitus is related to the peripheral auditory system,the other is related to the plasticity of auditory central system.In order to provide a new theoretical basis for the study and clinical treatment of tinnitus in the future,this paper studies the peripheral and central auditory system in the auditory pathway,and discusses the underlying pathogenesis of tinnitusNow,there have been many studies on tinnitus and peripheral auditory system,but there has been little research on the ribbon synapse,which is the first afferent synapse to transmit the sound information to the auditory nerve center.The transmission efficiency of synapses is not fixed.When synaptic neurons are excited,neurotransmitters are released.This process causes changes in postsynaptic potentials to achieve the goal of electrical signal transmission.We can find that the synapses have changed in number,function and morphology.During this change,synaptic transmission may be enhanced or weakened.These changes can be shorter?seconds to minutes?or longer?hours to weeks?.These changes are collectively referred to as synaptic plasticity.The changes of structure and function will affect the coding of sound,but there is little research on this aspect.Based on the above considerations,we intend to apply immunofluorescence double labeling method to the rat model of tinnitus induced by sodium salicylate.RIBEYE protein?pre-synaptic structure protein?and GluR2-3 protein?postsynaptic membrane receptor protein?were labeled respectively.The microstructures of ribbon synapses in inner hair cells were observed by transmission electron microscope before and after tinnitus.The changes of otoferlin protein were detected by Western blotting.In conclusion,the plasticity of the number,structure and function of ribbon synapses in the inner hair cells is studied in this study,which may be a new supplement to the study of the pathogenesis of tinnitus.At present,the main research on the plasticity of auditory cortex and tinnitus formation mechanism is that:auditory cortical plasticity refers to short-term or long-term secondary changes in neuronal excitability due to changes in synaptic afferent signals.These changes include changes in neuronal cell membranes?such as the opening and closing of ion channels?and synapses?for example,release?uptake and binding neurotransmitters?and the changes in nerve sensitivity and distribution.Moller et al.found in animal experiments that the changes of synaptic input signal can cause hypersensitivity in the auditory system when the hearing loss occurs in animals.If the auditory center misinterprets this as sound,it will produce tinnitus.Synaptic plasticity is regulated by a variety of mechanisms,including changes in the released neurotransmitters and transmission efficacy.On the contrary,the change of the amount of neurotransmitter receptors can also alter synaptic plasticity.Excitatory and inhibitory plasticity depend on the release of postsynaptic Ca²⁺levels,which is an important second messenger and plays a significant role in regulating cell metabolism.Ca²⁺signaling pathway manipulates cell growth,differentiation,and synaptic plasticity.Calmodulin?CaM?is the main Ca²⁺binding protein,and regulates basic functions of neurons.Ca²⁺/CaM dependent protein kinase II?CaMKII?is a key mediator of excitatory synaptic plasticity in center nerve system.However,the changes of Ca²⁺level in auditory cortex cells during tinnitus and the change of CaM/CaMKII have not been reported.In this study,we established tinnitus rat model by injecting sodium salicylate and investigated the auditory center-originated mechanism for tinnitus.Methods:72 Wistar rats with no external and middle ear diseases were selected.The rats were given by the Animal Experimental Center of China Medical University,at the age of 2.2-month-old,weighing 245±21.21 g.The rats were randomly divided into 3 parts,each part being divided into 4 groups with 6 rats in each group.Three of them were injected with sodium salicylate by intraperitoneal injection of 350mg/kg,respectively,and the control group was injected intraperitoneally with normal saline at the same time point.The cochlea basement membrane was separated by anaesthesia,and some of them were labeled with immunofluorescence,that is green fluorescence represented CtBP2 and red fluorescence represented postsynaptic membrane GluR2-3.The color of red and green fluorescence appeared in orange yellow at the same time.The significance of it is that there is a ribbon synapse.Under the confocal laser microscope,the basal membrane of the cochlea is scanned,and 3D reconstruction using 3Dmax8.0 software is used to analyze the changing process of the number of ribbon synapses in the inner hair cells,and some of them are observed under the transmission electron microscope.The microstructures of ribbon synapses before and after tinnitus were analyzed.The ribbon synapse is a dynamic structure which is constantly changing.Under the action of sodium salicylate,the different characteristics of the ribbon synapses microstructure are observed.In another part,Western blotting was used to detect the expression of otoferlin protein.Otoferlin protein played a role in the exocytosis of synaptic neurotransmitters.The expression of otoferlin protein changed at different time points of sodium salicylate.These changes can indirectly reflect synaptic function.Another 36 Wistar rats?provided by Animal experiment Center of China Medical University?were divided into 3 groups according to the principle of randomization,12 rats in each group:blank control group.normal saline group and tinnitus model group.The tinnitus model group injected by sodium salicylate?350 mg/kg?was tested by drinking water inhibition method.After the model was successfully established,the concentration of free Ca2+in auditory cortex was measured by Fura-2/AM fluorescence spectrophotometer.The expression and activity of CaM/CaMKII were detected by Western Blotting.Results:1.After 8 days of conditioned reflex training,the experimental rats established stable conditioned reflex.In the regression period of conditioned reflex,the rats in tinnitus group had the shorter time of conditioned reflex regression,and the statistical analysis showed that the difference among the two groups was statistically significant?P<0.05?.The tinnitus model was successfully made.2.The number of ribbon synapses in inner hair cells was found to be the highest on the 7th day after injection of sodium salicylate.Compared with the control group?P<0.05?,the number of ribbon synapses did not change significantly on the 3rd day after injection of sodium salicylate,but on the 10th day,there was a significant decrease in the number of ribbon synapses?P<0.05?.3.The ribbon synaptic structure of the inner hair nucleus in the subnuclear area can be seen clearly,including the typical structures,such as ribbon synapses,vesicles,synaptic spaces,and postsynaptic dense bands at different time points when sodium salicylate is injected.The ultrastructure and morphology of the ribbon synapses of the inner hair cells were also changed continuously.4.The expression of otoferlin was detected by Western blotting method at 140.5 KD.The expression of otoferlin protein was not very high before injection,and the expression of otoferlin changed continuously at each time point.At the third day of injection,the expression level began to increase,and on the seventh day,we could see that the expression level was the highest.On the 10th day,the expression of otoferlin protein in each group was significantly different?P<0.05?.5.After the establishment of the rat tinnitus behavioral model,the Ca2+concentration in the tinnitus model group increased significantly?P<0.05?,The expression of CaM in auditory cortex was also increased?P<0.05?and the activity of CaMKII was enhanced?P<0.05?.Conclusion:1.The method used in this study to study the number of ribbon synapses in the inner hair cells can solve the problem of not accurately calculating the number of synapses in the same plane because the ribbon synapses do not exist in the same plane.2.After injection of sodium salicylate,the number of ribbon synapses in the inner hair cells is constantly changing,beginning with a gradual increase in the number of synapses,and then decreasing,and increase or decrease the connection with the spiral neurons.It plays a compensatory role in tinnitus.3.The ribbon synapse of inner hair cell changes dynamically before and after sodium salicylate injection,and has different structural characteristics in different periods.The dynamic changes in the microstructure may affect the expression of the synaptic function.4.The expression of otoferlin protein also changed dynamically before and after sodium salicylate injection.The possible mechanism was that the otoferlin expression increased the fusion of synaptic vesicles and the release of synaptic transmitters decreased.The possible reasons of the decreased otoferlin expression are that the synaptic metabolism is affected,the original morphology and function can not be maintained.5.The increase of Ca2 concentration in auditory cortex of tinnitus rats and the activation of downstream CaM/CaMKII signal transduction pathway were involved in the occurrence of tinnitus induced by sodium salicylate.Abnormal electrical activity and synaptic plasticity in the auditory cortex may play an important role in the pathogenesis of tinnitus.