听皮层突触可塑性与耳鸣中枢化机制研究

Tinnitus is the most common disease in Otology, and extremely difficult for treatment in clinic. There is little evidence of physiological base of tinnitus either clinical observations or animal experiment, the precise mechanism still remains unclear. Sodium salicylate (aspirin) is a widely used as a treatment inflammation and chronic pain and it has been known which can induce reversible tinnitus, and hearing loss daily treatment with salicylate can also induce animals to develop tinnitus. Our previous studies demonstrated that long-term administration of salicylate enhanced the electromotility of OHC (outer hair cell), which increased the average spectrum of cochleo-neural electrophysiological activity (ASECA). Our recent studies showed the changes markedly, but reversibly, in the expression of the excitatory glutamate receptor subtype NR2B and immediate early genes Arg3.1, Egr-1 mRNA and protein levels, and also some changes in synaptic ultrastructure of neurons in auditory cortex. These data suggested that there are some plastic changes in auditory cortex of tinnitus animal. All of those provide evidence that the neural changes associated with tinnitus maybe become progressively "centralized" over time. However, whether the enhanced electrophysiological activity leads to enhancement of auditory central activity and / or induces to auditory central plasticity, still remain largely unclear. In this project, we establish the animal model of tinnitus by long-term salicylate administration, moreover, using animal behavioral and electrophysiological test methods to verify the presence of tinnitus in the animal. To assess the auditory cortex activities and NMDA & GABAA receptor activities of synapses by microPET (Positron emission tomography) molecular imaging technology, to observe the dynamic synchronous alterations of ASECA during the long-term salicylate administration process and after salicylate treatment ceased, and to find out the corresponding relationship between alterations of the auditory cortex activity and animal behavior (presence of tinnitus in the animal ) at the time，also to reveal the role of auditory center activity on mechanisms of tinnitus generation. Using transmission electron microscopy (TEM) and molecular biology technique to understand correspondence the ultrastructure of auditory cortex and the expression of mRNA & protein （NMDA and GABAA receptor, Syn, GAP-43, PSD-95 and CaMkII et al）. To elucidate the mechanism of tinnitus on auditory central plasticity (tinnitus centralization), all those will provide theoretical basis for pathophysiological mechanisms and clinical interventions of tinnitus.

水杨酸盐致耳鸣确切机制尚不清楚，项目前期研究发现长期注射水杨酸盐可引起OHC电能动性增强，继而产生耳蜗神经电生理活动增强，新近观察到听皮层突触超微结构和中枢可塑性相关即早基因表达出现显著改变，表明听皮层可能存在可塑性改变，提出了耳鸣中枢化机制假设。项目拟按照经典方法建立长期注射水杨酸盐诱导耳鸣动物模型，用动物行为和电生理学方法筛选耳鸣动物；采用microPET技术评估听皮层功能活动及其突触递质受体活性；通过免疫和透射电镜及分子生物学等方法，了解听皮层突触超微结构及检测可塑性相关蛋白和递质受体mRNA及蛋白表达量。动态同步观察耳鸣建模过程中ASECA变化、听皮层功能活动和递质受体活性改变与动物行为学（出现耳鸣）在时间相位上的对应关系，明确听皮层突触功能、结构和递质受体等改变的内在联系，阐明听皮层可塑性形成机制，验证耳鸣中枢化机制假设，为耳鸣产生病理生理机制以及耳鸣临床干预治疗提供理论依据。

耳鸣是最常见的耳科疾病，水杨酸盐诱导耳鸣模型并被广泛而成熟地应用于耳鸣研究。前期研究发现水杨酸盐引起OHC电能动性增强、蜗神经电生理活动增强，从而导致听觉中枢神经递质和蛋白表达的改变，出现活跃和代谢升高现象。那么外周活动改变是否引起听觉中枢神经突触可塑性改变值得进一步研究。.项目主要研究方向：听皮层突触可塑性与耳鸣中枢化机制，主要有以下发现：.（1）水杨酸急性和慢性注射均可使听皮层F18-FDG的SUV值显著升高。杏仁核及小脑的SUV值升高仅出现在急性注射组。表明急性注射能引起听觉相关脑区神经活跃度普遍升高，为一种应激反应，慢性水杨酸注射可能使得听觉中枢出现可塑性改变，加速耳鸣信号的形成。.（2）水杨酸慢性注射组听皮层等听觉相关脑区C11-FMZ SUV值均呈现显著升高，而恢复组SUV值亦明显高于对照组。推测GABAA受体的表达持续上调是中枢兴奋性增高和耳鸣样行为的代偿。.（3）慢性注射水杨酸盐组大鼠听皮层等听觉相关脑区神经突触数量明显增加，突触前膜囊泡增多，突触界面曲率增加，突触面积增大，活性区增加，每一活性区的长度增加，突触间隙明显增宽，突触后膜致密物质厚度增加。形态学上提示突触活动亢进。.（4）耳鸣大鼠听皮层即刻早反应基因Arg3.1 mRNA及其蛋白的表达水平随着长期水杨酸盐作用呈可逆性升高趋势，；而听皮层中Egr-1基因出现表达可逆性下调。此外听皮层中BDNF和p-CREB显著升高。表明听皮层神经元可塑性改变可能参与了耳鸣的形成和发展。.（5）慢性注射组大鼠听觉相关脑区的Glu与GABA含量均高于对照组而Glu/GABA比值在各组间无统计学差异。慢性注射组大鼠听觉相关脑区NMDA受体/GABA受体比值高于对照组。提示谷氨酸能和GABA能系统在水杨酸盐的作用下表现为谷氨酸能系统占优势的失平衡，听觉神经中枢兴奋性增高。.结论及意义：通过动态观察长期注射水杨酸盐和停止注射后整个耳鸣发展过程，发现听皮层突触形态变化、神经功能改变、相关蛋白表达差异、中枢兴奋/抑制失衡与耳鸣样行为在时间相位上存在着对应一致关系。在耳鸣形成过程中，听皮层突触发生可塑性改变，导致听皮层功能发生兴奋性改变，表现为耳鸣中枢化的现象。因此，我们推测中枢可塑性（尤其是听皮层突触可塑性）在耳鸣中枢化中起着非常重要的作用。本研究提示改变中枢突触可塑性（去耳鸣中枢化）可以成为耳鸣临床干预的有效途径。