| ObjectiveTinnitus is one of the common clinical symptoms in otology.It has a high incidence and makes people distress and even lose the ability of work.However,the neuronal mechanism of tinnitus is not fully understood.Inferior colliculus of midbrain is an important relay nucleus of auditory pathway.Too much excitability or decreased inhibition of local neuronal circuitry can cause the tinnitus.Sodium salicylate-caused tinnitus in human and animals has been reported frequently,thus,sodium salicylate has been used to make the tinnitus animal models.Sodium salicylate caused tinnitus has been known inhibited GAB A A receptor activity.Our latest research has found that sodium salicylate could enhance the excitability of NMDA receptors,and may contribute to the process of tinnitus pathology.We also found that acid-sensing ion channel 1a could up-regulate NMDA receptor activity.We thus ask whether inhibiting ASIC la can effectively reduce the occurrence of tinnitus and whether it can prevent and treat tinnitus related to NMDAR over-excitation caused by sodium salicylate?Using patch clamp technique,primary inferior colliculus neuron cultures,animal behavioral studies,ABR auditory examine,Western Blot and other methods to study the mechanism of tinnitus origination and try to find out ways of the prevention and the treatment the tinnitus.Methods1.Fresh brain sections containing inferior colliculus were prepared from C57BL/6 mice 2 weeks after birth.Pharmacological separation of NMDAR current and observation of the effect of sodium salicylate on NMDAR current in perfusate;The effect of extracellular fluid pH down to 6.0 on NMDAR current was observed;To observe the current of NMDAR at extracellular fluid pH 6.0 after the application of ASIC la inhibitor(Flurbiprofen).2.The inferior colliculus were cultured for 8-11 days in vitro.Hoechst 33342 staining was performed after different treatments and the neural survival rate and the level of LDH were evaluated.3.C57BL/6 male mice with normal hearing and behavioral parameters from 6 to 7 weeks after birth were injected intraperitoneally with sodium salicylate for 9 days to establish tinnitus model.The success of sodium salicylate model was evaluated by GPIAS.Before and after drug administration,mice in each group were assessed by elevated plus-maze,open field and forced swimming,and the auditory function of mice was assessed by ABR.4.After the experiment,mice were decapitated and their hypothalamus tissues were taken out.The expression and phosphorylation of NMDAR subunits in each group were detected by Western Blot.The level of ASIC la and the content of apoptotic related protein cleaved caspase-3 were detected.Results1.Inhibition of ASIC1a may limit the excitability of NMDA receptors.2.Inhibiting ASIC1a can prevent the excitatory neuronal death of cultured inferior colliculus.3.GPIAS test showed that high doses of sodium salicylate could reliably induce tinnitus-like behavior,whereas inhibiting NMDAR or ASIC1a channels could effectively prevent the production of tinnitus-like behavior of mice.4.Inhibiting NMDA receptor or ASIC1a channel can improve anxiety.and depression-like behavior in sodium salicylate treated mice.5.Inhibiting NMDA receptor or ASIC1a channel can effectively prevent hearing loss induced by sodium salicylate injected mice.6.Western Blot results showed that inhibition of ASIC1a decreased the expression of NR2 subunit of NMDA receptor in inferior colliculus induced by sodium salicylate,and decreased the phosphorylation levels of NR2A and NR2B;Inhibiting ASIC1a decreased the membrane expression level of ASIC1a in inferior colliculus induced by sodium salicylate;Inhibiting ASIC1a reduced the content of cleaved caspase-3 in inferior colliculus induced by sodium salicylate.ConclusionBy inhibiting the excitability of NMDA receptor down-regulated by ASIC1a,it can change the excitation-inhibition imbalance of inferior colliculus nucleus nerve circuit and prevent and treat tinnitus induced by sodium salicylate in mice. |
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